The general informations about the age, weght, height, smoking, fish consumption, and the overall safety condition of the industries are summarized in Table 1. The workers in the fluorescent lamps industries were mostly untrained and unaware about the hazardous effects of Hg. The workers protection measures were inadequate because the special protective booties and face shields were not provided to the workers. Routine medical examination was not provided. No air monitoring equipment’s were installed and most of the machines were not properly working and stay idle and there was no recycling unit for broken lamps. In the industries, the workers were only male with a mean age of 26.70 y; therefore no female was included in the control group. The height 5.28 ± 0.20 feet, weight 56.90 ± 10.50 kg and experience 5.35 ± 4.81 y of the exposed workers and control group was recorded. The total working time was 10 h per day in two shifts and one holiday in a week. About 70% of the workers were using gloves during work. Among selected workers, only 40% workers were fish consumers and 10% were using Hg based dental amalgam filling 2 years before giving the biological samples for this research therefore the amalgam filling was quite old (Table 1). All the data collected through questionnaire survey was correlated with Hg concentrations in biological samples of the workers.
Parameters Exposed workers Control
Parameters Exposed occupational workers Control
Age (y) 26.70 ± 6.72 29.90 ± 5.27 RBCs (µg/L) Me-Hg 27.70 ± 5.36 1.67 ± 0.86# Gender (male*) 40 40 I-Hg 3.32 ± 0.75 0.14 ± 0.07# Height (ft) 5.28 ± 0.20 5.31 ± 0.16 T-Hg 31.87 ± 5.96 1.85 ± 0.94# Weight (kg) 56.90 ± 10.50 55.10 ± 8.92 Plasma (µg/L) Me-Hg 3.50 ± 0.68 0.16 ± 0.08# Experience (y) 5.35 ± 4.81 Na I-Hg 10.60 ± 1.95 0.41 ± 0.21# Working (h) 8 Na T-Hg 15.10 ± 2.77 0.59 ± 0.30# Smoker (%) 60 55 Urine (µg/L) Me-Hg 13.50 ± 6.89 0.31 ± 0.23# Masks user (%) 0 Na I-Hg 137.50 ± 24.20 2.38 ± 1.14# Gloves user (%) 70 Na T-Hg 154.60 ± 28.00 2.81 ± 1.30# Goggles user (%) 0 Na Hair (µg/g) Me-Hg 3.21 ± 0.73 0.30 ± 0.18# Fish consumer (%) 40 Na I-Hg 0.31 ± 0.17 0.03 ± 0.02# Protective cloths 0 Na T-Hg 3.64 ± 0.84 0.33 ± 0.20# Hg used in tooth filling (%) 10 Nr Nails (µg/g) Me-Hg 5.52 ± 0.92 0.37 ± 0.20# I-Hg 0.60 ± 0.26 0.03 ± 0.02# T-Hg 6.30 ± 1.62 0.41 ± 0.22# Note. *Each value of mean ± SD represents standard deviation (n = 40). Na not applicable. Nr, not reported. #P < 0.001.
Table 1. The demographic profile and the concentrations of Me-Hg, I-Hg, and T-Hg in RBCs, plasma, urine, hair and nails of exposed workers in Fluorescent lamps industries and control subjects
The Hg concentration was (T-Hg, Me-Hg, and I-Hg) determined in all five biomarkers including RBCs, plasma, urine, hair and nails samples of Hg exposed workers in the industries and control individuals (Table 1) for the assessement of the distribution of Hg species in different body organs after exposure and the effect of the demographic characteristics (age, weight, experience, smoking, fish consumption) on Hg concentration on the biological samples of exposed workers. For the purpose, the age of the selected workers was divided into four groups ranging from 18–23, 24–29, 30–34, and 35–40 y, whereas the weight and experience of the workers was also categorized in to four groups ranging from 40–49, 50–59, 59–68, and 69–74 kg and 1–3, 4–7, 7–9, and 9–12 y respectively.
The mean concentrations of T-Hg in RBCs and plasma (31.87 µg/L, RBCs) (15.10 µg/L, Plasma) were found significantly (P < 0.001) higher in exposed workers (n = 40) as compared to the control subjects (n = 40) T-Hg (1.85 µg/L, RBCs), (0.59 µg/L, Plasma) (Table 1) and its level was found 6 times higher than its respective permissible limit (5.0 µg/L) . The percentage distribution of Hg species in RBCs comprised of 80%–90% of Me-Hg and 10%–20% of I-Hg, might be the reason of conversion of ionic Hg into Me-Hg by the anaerobic bacteria present in the RBCs (Table 1) [22, 23]. However, demethylation of Me-Hg occurs in RBCs which leads to the presence of I-Hg concentrations counting for 10%–20%. The workers exposed to the metallic Hg (Hg0) through inhalation, ingestion and dermal contact. The 80% absorption occurs through inhalation. The inhaled Hg0 oxidized to Hg+2 which attached to the carbon atom and form Me-Hg. Therefore workers exposed to Me-Hg through fish consumption or oxidation of metallic Hg into Me-Hg in the human blood which is then distributed to the whole body as reported earlier. Once absorbed in the body tissues Me-Hg can pass the blood brain barrier and converted into I-Hg and accumulated there. The ratio between Me-Hg and I-Hg in RBCs was found 9:1. Thus the blood can be used as a good indicator for Me-Hg concentration . As compared to RBCs an opposite % age between Me-Hg and I-Hg in plasma samples of the workers was observed which was 30%–40% of Me-Hg and 60%–70% of I-Hg level . The ratio calculated for Me-Hg and I-Hg in plasma was 1:2, therefore blood plasma can be a good indicator for both Me-Hg and I-Hg analysis .
The relationship of RBCs and plasma Hg concentrations with age and weight of the exposed workers and control observed no significant result (Figures 1A, 1B, 2A, 2B), as the workers of the 2nd group with age ranged from 24–29 y and weight range from 50–59 kg is showing an elevated level of T-Hg concentration as compared to the third group which observed random changes in the Hg concentration with age and weight. The linear regression model observed that the age and weight of the workers and control has shown a significant relation only with Me-Hg in RBCs (R2 = 0.65, P = 0.0012 for Me-Hg) (R2 = 0.86, P = 0.009 for control) and no significant relation with Hg in plasma.
Figure 1. The concentrations of Me-Hg, I-Hg, and T-Hg in RBCs with (A) age, (B) weight, (C) experience, (D) smoking and fish consumption of the exposed workers and control subjects. Similar letters indicate no-significant difference while different letters indicate significant differences when compared with the control group.
Figure 2. The concentrations of Me-Hg, I-Hg, and T-Hg in plasma with (A) age, (B) weight, (C) experience, (D) smoking and fish consumption of the exposed workers and control subjects. Similar letters indicate no-significant difference while different letters indicate significant differences when compared with the control group.
The long-term Hg exposure was found to be directly related with the elevated concentrations of the Hg species (Figure 1C) which was assessed by the relationship of the experience length and Hg concentrations in RBCs and plasma, through linear regression (Supplementary Figure S1 available in www.besjournal.com which observed that the Hg accumulated with the passage of time in the blood after exposure for long period of time . Smoking and fish consumption were also significantly (P = 0.001) increasing the Hg concentration only in the RBCs of smokers and fish consumers when compared with non-smokers and non-fish consumer in exposed workers (Figure 1D) [24-26], but all workers were using fish in little quantity (once in a month) . However in control, the smokers RBCs and plasma concentration (0.84 µg/L, 0.25 µg/L respectively) is higher as compared to non smokers RBCs and plasma concentration (0.57 µg/L, 0.17 µg/L). Similarly the fish consumers RBCs (0.64 µg/L) and plasma (0.43 µg/L) concentration was also found higher than non fish consumers (0.45 µg/L RBCs) (0.16 µg/L plasma) in control individuals.
Figure S1. The linear regression between experience and Hg concentration in the blood (RBCs, Plasma) of the workers.
The distributions of Me-Hg and I-Hg in the whole blood (RBCs and plasma) were calculated as 75%:25% respectively because maximum part of the blood is made up of RBCs [24,28,29]. This percentage also confirms the above mentioned results.
The urine samples were found 90% of I-Hg whereas a small amount of organic Hg may be found due to the reason of rupturing of RBCs after spending life span of 120 days in the blood which contains bounded organic Hg as reported earlier [7, 24]. The ratio calculated between Me-Hg and I-Hg in urine was 1:10 which makes it a good indicator for I-Hg determination .
The mean concentrations of T-Hg in urine was found significantly (P < 0.001) higher in exposed workers than control subjects (Table 1) and its level was 39 times higher than the permissible limit set by WHO (T-Hg 4 µg/L) for urine Hg . The linear regression model between the experience length of the workers and Hg concentration in urine indicated a direct relation for the T-Hg (R2 = 0.896, P = 0.0001) and I-Hg (R2 = 0.942, P = 0.0001) concentrations, while no significant relationship with Me-Hg (R2 = 0.5, P = 0.06) because the concentration of Me-Hg is very low in urine. The regression analysis of urine with age (R2 = 0.08, P = 0.001), weight (R2 = 0.47, P = 0.006) and fish consumption of the exposed workers observed a weak relation while no significant relation with smoking [25, 32]. In control, the urine Hg concentration with age (R2 = 0.84, P = 0.0012) and weight (R2 = 0.0002, P = 0.9) has shown significant relation while no effect with fish consumption and smoking. The reported half-life (t ½) of Hg in the urine is 25–41 days, lower than the half-life (90–120) of Hg in the blood, observed that I-Hg rapidly leaves the body than Me-Hg  because of increased rate of excretion through urine [24, 28, 29].
The workers with experience ranged from 9–12 y observed the highest concentration of T-Hg, Me-Hg and I-Hg in urine samples among the four selected groups and the concentration was 198.2, 12.16, 173.3 µg/L, respectively (Figure 3C).
Figure 3. The concentrations of Me-Hg, I-Hg and T-Hg in urine with (A) age, (B) weight, (C) experience, (D) smoking and fish consumption of the exposed workers and control subjects. Similar letters indicate no-significant difference while different letters indicate significant differences when compared with the control group.
It was observed from the study that the workers near exhaust machine and long time working experience have a high level of Hg in their urine and are more vulnerable as compared to other workers because the machine released all of the processed Hg in the industry as a smoke.
Regarding Hg in hair and nails, 80%–90% of Hg in workers hair and nails was Me-Hg  and its ratio with I-Hg was found 10:1 for both hair and nails. The reported half-life (t ½) of the hair Hg is 65 days with the range of 35-100 days and a small amount of I-Hg was also detected. Me-Hg is lipophilic in nature and strongly attached to the sulfhydryl groups of the protein which are abundantly present in the hair and nails [7, 34].
It was observed that the mean Hg concentrations in hair was determined significantly (P < 0.001) higher in exposed workers (T-Hg, 3.5) (Me-Hg, 3.2) (T-Hg, 0.3 µg/g) as compared to control 0.33, 0.3, and 0.03 µg/g, respectively (Table 1), and the Me-Hg in the hair samples was found 3 times higher than the permissible limit (less than 1 µg/g) set for hair Hg . Statistically strong relation was found between Me-Hg in the hair and experience of the workers while no significant relation was found between hair Hg and fish consumption in exposed workers. The age, weight and smoking have no significant effect on hair Hg concentrations which supports the findings reported in previous paper [35, 36]. However in control, the Hg concentration in hair observed a significant relation with both age, weight, fish consumption (R2 = 0.0002, P = 0.91) and no significant relation with smoking (Figure 4).
Figure 4. The concentrations of Me-Hg, I-Hg, and T-Hg in hair with (A) age, (B) weight, (C) experience, (D) smoking and fish consumption of the exposed workers and control subjects. Similar letters indicate no-significant difference while different letters indicate significant differences when compared with the control group.
The mean concentrations of T-Hg (6.3 µg/g), Me-Hg (5.52 µg/g) and I-Hg (0.6 µg/g) was examined in workers nails and was found 15 times higher Hg in nails than control and 2 times higher than the permissible limit set for nail Hg (less than 5 µg/L) [37, 38].
In comparison, the relationship between nails Hg concentration and demographic characteristics was found different from that of hair. The nails Hg has a significant relation with age (R2 = 0.674, P = 0.002), weight (R2 = 0.743, P = 0.0003), experience length (R2 = 0.869, P = 0.0002) and fish consumption in both exposed and control individuals . It observed that nails Hg is influenced by long term exposure and fish consumption because the Hg accumulated in the nails with time. The nails Hg assessment also indicate the earlier Hg exposure [36, 37] (Figure 5).
Figure 5. The concentrations of Me-Hg, I-Hg, and T-Hg in nails with (A) age, (B) weight, (C) experience, (D) smoking and fish consumption of the exposed workers and control subjects. Similar letters indicate no-significant difference while different letters indicate significant differences when compared with the control group.
Hg Concentration in RBCs and Plasma
Hg Concentration in Urine
Hg Concentrations in Hair and Nails
This is a twofold study in which the first part was focusing on the assessment that wether the Hg exposure and the concentration found in the biological samples can cause health problems among workers, and the second was to investigate that what pattern has the Hg and its species adapt when get entered into the body. The study observed that the industrial exposed workers exhibited significantly (P = 0.001) higher concentrations of Hg in their blood (RBCs, plasma), urine, hair and nails samples as compared to control as well as the concentration was found several times above the permissible limit set for all biological samples. The relationship of Hg concentration between biological samples and demographic characteristics (age, weight, experience, smoking, fish consumption) of both Hg exposed workers and individuals selected as control was assessed by linear regression observed that Hg in RBCs, plasma, urine, hair and nails has a significant relation with experience of the workers  while a weak relation with age, weight, smoking and fish consumption [25, 26]. However in control a strong relation was observed for fish consumption and weak relation with age and weight .
The highest Hg concentration was found in the biological samples of four workers aged 26, 29, 35, and 40 y. Those workers were working in the exhaust machine from long time with experience of 12 and 14 y. This data showed that workers working near the vicinity of exhaust machine are more vulnerable to the toxic Hg as compared to the basing and sealing machine. This study also unveiled that the age and weight were found independent variables for the assessment of Hg concentration  while exposure time and vulnerability greatly influence the Hg concentration in the biological samples.
According to the WHO report Hg is considered as one of the top ten toxic chemicals of public health concern greatly affect immunity, digestive system, kidneys and lungs. Exposure for some hours can lead to headache, cough, chills, shortness of breathing and metallic taste etc. The concentration found in different biological samples not only bioaccumulate in different body organs but this can be the reason of many neurological and kidney diseases , because the concentration above the permissible limit is alarming for the workers under study. The different diseases noted through questionnaire survey among the workers showed that 20% of the workers complaining the symptoms of insomnia and loss of appetite while 30% reported memory disturbances because Hg when passed to the brain, it change its forms and accumulate there causing neurological diseases including hand and eyelid tremor, memory disturbance and insomnia which supports the previous studies conducted so far . In the study area, 35% workers complained headache and body aches with fatigue and two workers were reported with mental health problems, the detail of which has been given in Supplementary Table S1 (available in www.besjournal.com). A previous case study  reported nephrotic syndrome in the workers of fluorescent tube recycling factory. The concentration noted was 41 µg/L in the blood and the protein excretion in urine was 12.3 g/d which then reached to 0.32 g/d after withdrawl from Hg. However, this is rarely occurs because membranous glomerulonephritis would never develop after chronic Hg exposure. The urinary problems regarding burning micturition, some often blood seen in urine was reported in 26% of exposed workers working near exhaust machine which is the effect of high concentration Hg exposure .
Parameters Exposed Control Mean Mean Age (yr) 26.7 ± 6.7 29.9 Gender Male Male Height (ft) 5.28 ± 0.20 5.31 ± 0.3 Weight (Kg) 56.9 ± 10.5 55.1 ± 11.2 Vacation on Occasion on Occasion Experience (yr) 5.35 ± 4.81 6.21 ± 4.51 Working (hr) 10 10 Masks user/non user (%) 0 0 Goggles user/non user (%) 0 0 Dental amalgam user (%) 10 30 Gloves user/non user (%) 70/30 70/30 Smokers/nonsmokers (%) 60/40 55/45 Fish consumer/non consumer (%) 40/60 50/50 Urinary problems (%) 26 5 Headache (%) 35 10 Memory disturbances (%) 30 5 Loss of appetite (%) 20 5 Night blindness (%) 1 NRa Insomnia (%) 20 NR Fatigue (%) 33 5 Body aches (%) 35 10 Finger and eyelid tremor (%) 20 NR Tiredness (%) 67 20 Two workers with MHP (mental health problem) Note. a Not Reported.
Table S1. Results of questionnaire regarding diseases prevailing among the selected workers
The results of this study observed that the Hg distributed in the whole body but the species of Hg (Me-Hg, I-Hg) are showing different pattern of distribution among the body organs. The Me-Hg was found dominant specie in the RBCs, hair and nails samples [24, 29] while I-Hg was found dominant specie in the urine and plasma samples .
The current study results were compared with other reported literature as shown in Table 2. The Hg concentration found in the previous studies [4, 51] conducted among the workers of flurecsent lamp recycling factory, goldmining workers and goldsmith had a higher blood Hg concentration similar to the current study results. The total Hg concentration was found much lower in studies on children for IQ test  and in dental amalgam users . In comparison with the current study the highest concentration of Hg was found in a study which analyzed Hg (590–1,099 μg/g) in workers nails [52-55].
No Parameters Current study Reported literature Concentration Mean Remarks Country Reference 1 Urine 154.6 ± 28 μg/L 1,060 μg/g creatinine In workers of small scale goldmining Wuchuan, Guizhou, China  2 Urine 13.85 ± 13.14 µg/g creatinine Workers of compact fluorescent lamp factory Iran  3 Urine 1.34 μg/L Dental amalgam users USA  4 Urine 9.4 μg/L In children living near high Hg contaminated artisanal gold mining area Nangaritza River Basin, Ecuadorian Amazon  5 Hair 3.64 ± 0.84 μg/g 0.53 μg/g Cinnabar mining region Huancavelica, Peru  6 Hair 2.4 μg/g Cinnabar mining region Almaden, Spain  7 Hair 69.3 μg/g In workers of small scale goldmining Wuchuan, Guizhou, China  8 Hair 3.8 μg/g Gold minning region Bolivia  9 Fingernails and toenails respectively 6.3 ± 1.62 μg/g 590 μg/g and
Workers in chloralkali plant Punjab Pakistan  10 Fingernails 0.14–27.27 mg/kg In operative dentists Tehran  11 RBCs 31.87 ± 5.96 μg/L 33 ± 8.8 μg/L Goldsmith workers Pakistan  12 Plasma 15.1 ± 2.77 μg/L 11.8 ± 3.2 μg/L Goldsmith workers Pakistan  13 Blood 46.9 μg/L 26.8 ± 14.6 μg/L Goldmining workers Suriname  14 Blood 1.61 μg/L In children for IQ test Southern China  15 Blood 38 μg/L Workers in Fluorescent lamp recycling facility Wisconsin 
Table 2. Comparison of Hg concentration in biological samples with reported literature
The study concluded that workers in the industries are vulnerable to the toxic level of Hg and they must be provided with all protective clothing, gloves, shoes and proper duty rotation which ensure their protection from Hg. To protect workers exposure through breathing, the respirator properly fitted, tested with mercury vapor cartridge and the manufacturer should regularly monitor the health of the individuals and precautionary measures should be taken to avoid any serious problems. The Environmental Protection Agency (EPA) of the Pakistan has to strictly implement the laws and regulations regarding handling of toxic metals and regularly monitor the industries to ensure safe and protected environment for workers and general public living near surroundings.