ดูโค้ด

RISK ASSESSMENT OF HEAVY METAL ASSOCIATED WITH DERMAL EXPOSURE IN INCENSE WORKERS IN SMALL HOUSEHOLD FACTORIES AT ROI-ET PROVINCE, THAILAND
Pornrat Kaewrueng1, 2, 3, Wattasit Siriwong4, 5
1International Postgraduate Programs in Environmental Management, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand, 2National Center of Excellence for Environmental and Hazardous Waste Managemet (NCE-EHWM), 
Chulalongkorn University, Bangkok 10330, Thailand, 3 phornrat.k@gamil.com, + 66 (0) 8 0449 9955, 4College of Public Health Science, Chulalongkorn University, Bangkok 10330, Thailand, 5Thai Fogarty ITREOH Center, Chulalongkorn University, Bangkok 10330, Thailand, corresponding: wattasit.s@chula.ac.th, +66 2 218 8182.

ABSTRACT:  The Ngooleum village in Roi Et province is one of the largest producers of incense stick in the northeastern region of Thailand. An exposure of heavy metal through dermal contact is concerned because packaging is the main production process with the main reason to concern is on their usage of less quality of dyeing color that may contain some heavy metals. The aim of this study was to assess the potential risk of heavy metals related to dermal exposure of incense workers. Thirty-five small household factories were selected randomly to collect heavy metal residues on one packaging worker’s hands of each factory. Heavy metals residue in each sample was extracted by microwave digestion method and analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES). The average residue concentrations (±SD) of Ba, Cd, Cr, Mn, Ni, and Pb on worker’s hands were 11.03±2.31 mg/kg, 1.13±0.23 mg/kg, 2.77±0.83 mg/kg, 7.06±1.92 mg/kg, 8.20±2.22 mg/kg, and 3.55±1.32 mg/kg, respectively. The dermal absorbed dose (DAD) of female workers (n=30) of Ba, Cd, Cr, Mn, Ni, and Pb were 2.58×10-7 mg/kg-day, 2.68×10-9 mg/kg-day, 6.39×10-8 mg/kg-day, 1.66×10-7 mg/kg-day, 1.95×10-7 mg/kg-day, and 8.28×10-8 mg/kg-day, respectively. The risk in term of hazard quotient (HQ) of Ba, Cd, Cr, Mn, Ni, and Pb were 5.26×10-5,   2.24×10-4, 4.26×10-3, 2.77×10-4, 9.74×10-4, and 1.97×10-4, respectively and hazard index (HI) for six heavy metals was 5.99×10-3. Another dermal absorbed dose (DAD) from male workers (n=5) of Ba, Cd, Cr, Mn, Ni, and Pb on worker’s hands were 2.77×10-7 mg/kg-day, 2.64×10-9 mg/kg-day, 7.38×10-8 mg/kg-day, 1.71×10-7 mg/kg-day, 1.89×10-7 mg/kg-day, and 8.98×10-8 mg/kg-day, respectively. The hazard quotient (HQ) of Ba, Cd, Cr, Mn, Ni, and Pb were 5.64×10-5, 2.20×10-4, 4.92×10-3 , 2.85×10-4, 9.46×10-4, and 2.14×10-4, respectively and the hazard index (HI) was 5.56×10-3. In conclusion, both of the HQ values for single heavy metal and HI value for six heavy metals were greatly below one indicated incense workers may not be greatly risk from these heavy metals via dermal route.
Keywords:  Heavy metal, Dermal exposure, Risk assessment, Incense worker

1. Introduction
	Incense stick has been widely used especially in Asian countries, which respect to Buddhism, Taoism and Shinto religions [1]. The reasons why incense stick are used because each religion believes to use it for connection with god in many holy ceremonies or it use for sending soul of the deceased. Thailand is one of Asian country that adopt the believing from India that use and produce incense stick in several provinces such as Ayutthaya, Chiang Mai, Chai nat, Singburi, Pathumthani, Uthaithani and Roi Et. In this case study, Roi Et is chosen because it is one of the largest production of incense stick in the northeastern region of Thailand  and  has many small household factories. Ngooleum village, Dong dang sub district, Chaturaphak phiman district in Roi Et province has 12 villages, so Moo 12 was selected because it can earn the best income from incense for a living more than other villages. The main occupation in the village is incense worker; other occupations are farmer, shallot growing, merchant, and employee for supporting income.  This area produces many types of incense stick but incense stick that Thai people call “ Toop-sad” is the most popular product. Its length  is about 20 centimeters, the top of bundle is mold with saw dust and other components, so the bottom is always colored by dipping in red and pink colors. Incense stick process can be separated into 6 steps, which are bamboo preparation, incense powder mixing, incense molding, color dipping and dyeing, perfume spraying, and packing. All of incense making steps, the packing step has the highest risk to contact dyeing color because workers pack incense without hand protection.  The dyeing color in incense making process is synthetic color powder that may compose of heavy metals such as lead, cadmium, and zinc. Heavy metals can cause the problem in long term, which the main effects are cancer, kidney damage, nervous system damage, skin changes, impaired cognitive, lethargy, insomnia, and emotional instability, however, the signs can occur in acute effect as headaches, skin rash, cramp, vomit, sweat and inconvenient breathing [2].  Dermal exposure is the main route that the dyeing colors contact human body so hand wipe method is employed to collect samples of heavy metals on workers’ hands. 
2. Site description
	Roi Et is located northeastern region of Thailand. The area is divided into 20 districts, so the districts are subdivided into 193 subdistricts  and 2,311 villages. Ngooleum, which is a village in Dongdand subdistrict, Chaturaphak phiman district. The village has total area of 2,380 rai which separated into 200 rai of residence and 2,180 rai of workplace [3].
	In the case study, Moo 12 of Ngooleum village is selected because it earns the best income from incense small household factory and incense worker is the main occupation of villagers. 
3. Methodology
3.1 Sampling
	In the study area, there are around 100 small household factories so thirty-five small household factories of those were chosen for sample collection [4]. Dissolved dye, incense stick, and hand wipe samples were collected from each small household factory.  All of incense making process, the workers who responsible to packing incense were chosen randomly to collect samples by gauze wiping technique. 20 ml of dissolved dye samples were collected and kept in plastic bottles as well as one pack of incense stick samples were collected in plastic bags per one small household factory. 
	Gauze wiping technique, use of two 20% isopropanol gauze pads wipe the hand surface [5], side to side in “Z” wipe pattern both of vertical and horizon axial, including front and back sides and applying to wipe fingers  [6] 10 seconds per hand. Daily working day, hand wipe samples of each worker were collected three times per small household factory: in the morning before incense process, at lunch when workers finish work, and when after workers wash their hands before having lunch. Only ten of thirty-five small household factories were collected samples in four times, so the samples that workers use gloves will be collected in the afternoon when workers finish work.
	A questionnaire for surveying dermal exposure of heavy metal is divided into three parts. Thirty -five participants are chosen randomly from each household factory for wiping and question them face-to-face in the morning before workers start daily work. In addition, the questionnaire is used to ask other sixty-five workers as 100 workers totally including thirty-five wipe subjects. For sixty-five general workers will be randomly selected 1 or 2 workers from each small household factory. The questionnaire includes age, gender, individual exposure parameters, and subjective sign and symptom of dermal contact.
3.2 Chemical analysis		
	 Microwave digester was used for sample digestion, so microwave methods of each sample were different [7]. The digested samples were measured six heavy metals concentrations as barium (Ba), cadmium (Cd), chromium (Cr), manganese (Mn), nickel (Ni), and lead (Pb) by using inductively coupled plasma optical emission spectrometry VISTA-MPX Axial (ICP-OES). Quality controls of the samples present limit of detection (LOD), method recovery, and relative standard deviations (RSDs). The LOD of Ba, Cd, Cr, Mn, and Ni of dissolved dye, incense stick, and gauze wiping pad were 5.0x10-3, except Pb was 1.0x10-3. The reasonable method recoveries of those heavy metals in dissolved dye, incense stick, and gauze-wiping pad were in the range of 85.3 to 97.3%, 85.5 to 99.7%, and 89.4 to 102.6%, respectively. The RSDs of dissolved dye, incense stick, and gauze-wiping pad of those heavy metals in the range of 3.8 to 9.3%, 4.1 to 9.0%, and 5.4 to 8.9%, respectively.
3.3 Risk assessment 
	Risk assessment is the process that estimates the probability of risk and negative health effect in human who is exposed to chemical substances. The risk assessment consists of four basic steps  (1) hazard identification (2) dose response assessment (3) exposure assessment, and (4) risk characterization [8], [9]. In this study, the objective of hazard identification step was to identify six heavy metals as barium (Ba), cadmium (Cd), chromium (Cr), manganese (Mn), nickel (Ni), and lead (Pb), so all of them were concerned about their non-carcinogenic effects. The dose response assessment was done for explaining the relationship between non-carcinogenic effect and the amount of those heavy metals in step 1 in term of dermal reference dose (RfDd). The RfDd of Ba, Cd, Cr, Mn, Ni, and Pb were 4.90 x10-3, 1.20 x10-5, 1.50 x10-5, 6.00 x10-4, 2.00 x10-4, and 4.20 x10-4 mg/kg-day, respectively. The main route of the exposure is dermal contact, so the exposure assessment step use dermal absorbed dose (DAD) formula (see Equation 1,2,3) to calculate dose in incense workers through dermal route. The value of each parameter in the DAD and DAevent formulas are presented in Table 1. The formulas are as follow;

DAD =  (DAevent x SA x EV x ED x EF)        (1)
                            (BW x AT)
DAevent = C x CF x AF x ABS	           	 (2)
SA	= a0Wa2Ha1 			(3)

Risk characterization step in the case study, the toxicity was classified as non-carcinogen. The potential risk is compared between dermal absorbed dose (DAD) and reference dose (RfDd) in hazard quotient (HQ) formula for single substance (see Equation 4). For multiple substance or multiple routes, use hazard index (HI) to calculate the non-carcinogenic risk (see Equation 5). If HQ equation more than 1 that mean the exposure more than reference dose, the non-carcinogenic risk should be concerned [10].

HQ = DAD                  		(4)	
            RfDd
HI = Σ HQ			(5)


Table 1 Description and value of each parameter in the formulas
Parameters	Description	Unit	Value
 	 	 	Male (n=5)	Female (n=30)
SA	Surface area	cm2	903.1	803.0
DAevent	Absorbed dose per event	mg/cm2-event	Chemical-specific
EV	Event frequency	event/day	1.8	1.7
ED	Exposure duration	years	19.0	18.7
EF	Exposure frequency	day/year	365	365
BW	Body weight	kg	64.2	58.3
AT	Averaging time for non carcinogen	days	365	365
C	Heavy metal concentration on both hands	mg/kg	-	-
CF	Conversion factor	kg/mg	10-6	10-6
AF	Adherence factor of soil to skin (US EPA, 1997b)	(mg/cm2- event)	0.1	0.1
ABS	Dermal absorption fraction	-	      0.01, 0.001* 
H	Height	cm	167.2	156.0
W	Weight	kg	64.2	58.3
a0	Constant values (US EPA, 1997)	-	0.0257	0.0131
a1	Constant values (US EPA, 1997)	-	0.573	0.412
a2	Constant values (US EPA, 1997)	-	-0.218	0.0274
* only Cd
4. Results and discussion
4.1 Heavy metal concentrations in dissolved dye, incense stick, and hand wipe samples on workers’ hands 
	In dissolved dye, the average concentration (±SD) of the six heavy metals as Ba, Cd, Cr, Mn, Ni, and Pb were 1.47±0.21 mg/L, 0.16±0.01 mg/L, 1.34±0.13 mg/L, 1.12±0.10 mg/L, 0.64±0.08 mg/L, and 0.90±0.05 mg/L, respectively. The average concentration (±SD) of those heavy metals in incense stick were 1.30±0.29 mg/kg, 0.08±0.03 mg/kg, 0.89±0.10 mg/kg, 0.87±0.13 mg/kg, 0.99±0.19 mg/kg, and 0.27±0.05 mg/kg, respectively. The average concentration (±SD) of Ba, Cd, Cr, Mn, Ni, and Pb on workers’ hands were 11.03±2.31 mg/kg, 1.13±0.23 mg/kg, 2.77±0.83 mg/kg, 7.06±1.92 mg/kg, 8.20±2.22 mg/kg, and 3.55±1.32 mg/kg, respectively.
	Post hoc LSD was used to compare the average heavy metal concentrations in paired sample between dissolved dye and incense stick, dissolved dye and hand wiping sample, and incense stick and hand wiping sample. The results showed the mean difference of Ba, Mn, and Ni between dissolved dye and incense stick were not statistically significant (p=0.597, 0.339, and 0.261, respectively), but Cd, Cr, and Pb were statistically significant (p=0.000) (see in Table 4.9). The mean difference of Ba, Cd, Cr, Mn, Ni, and Pb for paired between dissolved dye and hand wiping sample were statistically significant (p=0.000) and incense stick and hand wiping sample were statistically significant (p=0.000). In Figure 1 showed the bar graph of the average concentrations for dissolved dye, incense stick, and hand wiping samples with the different letter are significantly different at p≤0.05. When compared the mean heavy metal concentrations of dissolved dye and incense stick with hand wiping sample, the results found that were statistically significant because surface area of hands more than dissolved dye and incense stick. Another reason was during working incense process around 3-4 hours per event had accumulated dyeing color on hands. 

 
(a,b,c : The mean concentrations with the different letter are significantly different at p≤0.05)
Figure 1 Heavy metals concentration of dissolved dye, incense stick, and on workers’ hands
4.2 Heavy metal concentrations on workers’ hands during working
	Thirty-five workers were separated into two groups. Twenty-five workers in the first group were collected three times of hand wiping samples as before working, after working, and after washing hands. Only ten workers in the second group were collected hand wiping samples four times, so after working by using gloves was added. In Table 2, Willcoxon test was used to compare the average concentration of heavy metal between two groups. Using Willcoxon test  of before and after working, the result was statistically significant difference (p=0.000) for all of heavy metals and the percentage of heavy metal concentration increasing in the range of 95.33-97.75%.  After working and after washing hands was statistically significant difference (p=0.000) for all heavy metals of Willcoxon test, so the percentage of heavy metal concentration decreasing in the range of 88.09-97.18%. Using Willcoxon test  of before working and after washing hands, the mean difference of all heavy metals were statistically significant (p=0.000) except Pb (p=0.027) and the percentage of heavy metal concentration that remaining in the range of 15.38-68.18%. The last paired was tested because in the questionnaire indicated all participants did not use gloves when working. Thus after working by non-using gloves and using gloves were compared and the results showed that average concentration of all heavy metals were statistically significant difference (p=0.000), so the percentage of heavy metal concentration that decreasing in the range of 97.11-99.76%. 
Table 2 The average heavy metals concentration during working
Elements	Average concentration  (±SD) (mg/kg)
	Before working 
(n=35)	After working 
(n=35)	After washing hand   
(n=35)	Use gloves (n=10)
Ba	0.28 ± 0.15	11.03 ± 2.31	0.88 ± 0.39	0.08 ± 0.03
Cd	0.06 ± 0.04	1.13 ± 0.23	0.10 ± 0.06	0.02 ± 0.00
Cr	0.07 ± 0.05	2.77 ± 0.83	0.33 ± 0.15	0.08 ± 0.04
Mn	0.33 ±0.17	7.06 ± 1.92	0.48 ± 0.17	0.14 ± 0.01
Ni	0.33 ±0.18	8.20 ± 2.22	0.39 ± 0.17	0.02 ± 0.01
Pb	0.08 ± 0.05	3.55 ± 1.32	0.10 ± 0.04	0.03 ± 0.01

4.3 Risk assessment
	The table 3 was presented value of DAD, RfD, HQ, and HI as well as average mean, and RME at 95th percentile. The results showed that HI results of mean for male were 5.65×10-3. HI of mean and RME in female were 5.99×10-3  and 2.79×10-2 and results of total thirty-five workers were 6.02×10-3 and 2.84×10-2 , respectively.
	Both of HQ and HI results as well as average mean and RME were less than one that means the potential risk of this case study was no non-carcinogenic risk.
Table 3 Summary of dermal exposure and risk values of incense workers
 	Elements	DAD	RfDd	HQ	HI
 		Mean	RME	 	Mean	RME	Mean	RME
Male	Ba	2.77 x10-7	NC	4.90 x10-3	4.94 x10-5	NC	5.65 x10-3	NC
	Cd	2.64 x10-9	NC	1.20 x10-5	2.06 x10-4	NC		
	Cr	7.38 x10-8	NC	1.50 x10-5	4.05 x10-3	NC		
	Mn	1.71 x10-7	NC	6.00 x10-4	2.58 x10-4	NC		
	Ni	1.89 x10-7	NC	2.00 x10-4	8.99 x10-4	NC		
 	Pb	8.98 x10-8	NC	4.20 x10-4	1.85 x10-4	NC		
Female	Ba	2.58 x10-7	1.02 x10-6	4.90 x10-3	5.26 x10-5	2.09 x10-4	5.99 x10-3	2.79 x10-2
	Cd	2.68 x10-9	1.06 x10-8	1.20 x10-5	2.24 x10-4	8.68 x10-4		
	Cr	6.39 x10-8	3.20 x10-7	1.50 x10-5	4.26 x10-3	2.02 x10-2		
	Mn	1.66 x10-7	7.00 x10-7	6.00 x10-4	2.77 x10-4	1.17 x10-3		
	Ni	1.95 x10-7	8.87 x10-7	2.00 x10-4	9.74 x10-4	4.36 x10-3		
 	Pb	8.28 x10-8	4.68 x10-7	4.20 x10-4	1.97 x10-4	1.06 x10-3		
Total	Ba	2.58 x10-7	1.00 x10-6	4.90 x10-3	5.26 x10-5	2.04 x10-4	6.02 x10-3	2.84 x10-2
	Cd	2.64 x10-9	1.04 x10-8	1.20 x10-5	2.20 x10-4	8.64 x10-4		
	Cr	6.48 x10-8	3.12 x10-7	1.50 x10-5	4.32 x10-3	2.08 x10-2		
	Mn	1.65 x10-7	6.83 x10-7	6.00 x10-4	2.75 x10-4	1.14 x10-3		
	Ni	1.92 x10-7	8.66 x10-7	2.00 x10-4	9.59 x10-4	4.33 x10-3		
 	Pb	8.30 x10-8	4.57 x10-7	4.20 x10-4	1.98 x10-4	1.09 x10-3		
NC = not calculated


4.4 Subjective sign and symptoms
	Although almost participants had nothing in general health, but allergy, migraine, gastritis, hypertension, liver disease, kidney disease, and lymph node cancer were reported from some participants. Hand eczema, skin rash, cramp, vomit, sweat, inconvenient breathing, headache are general health symptoms that can occur when workers expose to heavy metals, so that always found about the report or from the participants’ symptom in sweat and headache. Participants were found rarely and nothing in hand eczema, skin rash, cramp, vomit, sweat, inconvenient breathing, and headache. The general health symptoms are effects from any route that expose to heavy metals but there are few data of health symptoms from heavy metals [11], [12], [13], and [14]. 
Table 4 Subjective health and symptoms in Moo 12 of  Ngooleum village in Roi Et province, Thailand
Characteristics	Incense workers
	Male	Female
	(n=65)	%	(n=35)	%
Health symptoms				
Allergy	1	1.5	0	0
Migraine	0	0	3	8.6
Gastritis	2	3.1	0	0
Hypertension	0	0	1	2.9
Liver disease	2	3.1	0	0
Kidney disease	1	1.5	1	2.9
Nothing	59	90.8	29	82.9
Lymph node cancer	0	0	1	2.9
Symptoms from working				
Hand eczema				
Always	0	0	0	0
Rarely	2	3.1	4	11.4
Never	63	96.9	31	88.6
Skin rash				
Always	0	0	0	0
Rarely	8	12.3	9	25.7
Never	57	87.7	26	74.3
Cramp				
Always	0	0	0	0
Rarely	10	15.4	11	31.4
Never	55	84.6	24	68.6
Vomit				
Always	0	0	0	0
Rarely	2	3.1	2	5.7
Never	63	96.9	33	94.3
Sweat				
Always	8	12.3	2	5.7
Rarely	31	47.7	13	37.1
Never	26	40.0	20	57.1
Inconvenient breathing				
Always	0	0	0	0
Rarely	14	21.5	2	5.7
Never	51	78.5	33	94.3
Headache				
Always	0	0	1	2.9
Rarely	9	13.8	14	40.0
Never	56	86.2	20	57.1


5. Conclusions
	When compared heavy metal concentrations in dissolved dye and incense stick with hand wiping samples, the results found statistically significant. For all paired samples, the  Willcoxon test were statistically signignificant that mean heavy metal concentrations in each event of collecting were different clearly. Thus incense workers should wash their hands after working and use gloves during working to reduce heavy metal concentrations on hands. However, the HI results of male, female, and total were less than one as well as average mean and RME. Thus incense workers may not be greatly risk from these heavy metals via dermal route. Allergy, migraine, gastritis, hypertension, liver disease, kidney disease, and lymph node cancer were found in participants. Hand eczema, skin rash, cramp, vomit, sweat, inconvenient breathing, headache were almost found rarely in participants.

Acknowledgement
	I would like to express my financial support from The National Center of Excellence for Environmental and Hazardous Waste Management (NCE-EHWM) and scholarship of Graduate school from Chulalongkorn University. I also would like my gratitude to Thai Fogarty ITREOH Center (Grant Number: D43 TW007849 Fogarty International Center National Institutes of Health).

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