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Background
In recent years, cosmetic industry has experienced a significant progress, including the production of various types of cosmetics, such as soaps, creams, face powder, tanning lotions, toothpaste, and lipsticks, for the skin, hair, teeth, body, and nails care (1). Based on previous research, the consumption of cosmetic products is very common in Iran. According to the statistics, Iran and Saudi Arabia have the highest cosmetic consumption rate in the Middle East; in this regard, about $ 2.1 million is spent on cosmetic products in Iran (2). Hair color is one of the best-selling products since about 25% of women aged above 18 years and 10% of men aged over 40 years use hair color in Europe, North America, and Japan (3).
Hair colors include ammonium compounds (e.g., 4-amino2-hydroxytoluene and amino phenol), antioxidants, ammonia, soap, aroma compounds, moisturizers, forming creams, alkalisers, and metal oxides (e.g., titanium dioxide and iron oxide) (4) Some of the substances like heavy metals in cosmetics are unwanted or produced during the production process (5). Heavy metals are among the most important environmental pollutants, many of which are toxic to humans even in small quantities (6). These kinds of metals are capable of producing free radicals and active oxygen species and damaging the cell cycle, cellular organelles, and their activities (7). The persistent usage of polluted products results in the accumulation of non-metabolized toxins in the target tissue, thereby leading to the incidence of several diseases and complications (8).
With regard to the high prevalence of using cosmetics in Iran, the present study was conducted to determine the concentration of three heavy metals, namely lead, chromium, and cadmium, in the most popular chemical hair color brands available in the market of Qom, Iran.
 
 

Materials & Methods

 
Sampling Method
This cross-sectional descriptive-analytical study was conducted in Qom in 2017. Information about the most commonly used hair color brands was gathered by interviewing and distributing questionnaires among famous makeup sale representatives, hairdressers, and women aged above 40 years. The last-mentioned group was selected because of their persistent and frequent use of hair colors as a result of white hair and lack of natural pigments. After reviewing 12 brands (i.e., 7 and 5 domestic and foreign brands, respectively), 3 samples from each brand were randomly purchased from the most popular stores located in four areas of Qom. The samples were prepared in the laboratory using digestion method and then the levels of lead, cadmium, and chromium were measured by means of the atomic absorption spectrometer (AAS).
 
Sample preparation method
The extraction of the heavy metals was accomplished using the dry and wet methods, as well as microwave digestion system. In the dry method, 10 g of the sample was weighed in a crucible and then placed in the oven at 100ºC until reaching a constant weight. In the next stage, for the ash preparation procedure, the crucible was first placed in a furnace at 200ºC. Subsequently, the furnace was heated to 400ºC, and its temperature was added 50ºC per hour. The solution was kept at this temperature for 8 h. Afterward, 5 ml of 6 M hydrochloric acid was added to the crucible so that all the ashes could be exposed to acid.
The solution was heated until evaporation, and the resultant sediments were dissolved in 10-30 ml nitric acid 0.10N-standardized. The crucibles were carefully centrifuged so that all the ashes would be in contact with the acid. Then, the solution was covered by a plastic cover and left intact for 1-2 h. In the next stage, deionized water was added to the solution until its volume reached 25 cc (9).
To prepare the samples through the wet method, 5 ml of nitric acid 0.10N-standardized was added to 1 g of the sample. Then, the samples were placed on a heater at 85º C for 4 h to extract the yellow vapors. After reaching the room temperature, 1 ml of 0.0030 oxygenated water was added to the solution and then passed through Whatman filter paper. Subsequently, deionized water was added to the solution until obtaining a volume of 25 cc. Finally, the absorption rate was measured by graphite furnace AAS (GFAAS) (10).
In order to investigate the influence of the acid types, used in sample preparation stages, on the adsorption rate, measured by GFAAS, one of the samples that had the highest concentration of heavy metals (i.e., lead, cadmium, and chromium) was chosen to be subjected to digestion and preparation procedures using different acids. The methods are explained below.
To this end, five crucibles containing 1 g of the sample were dried in the oven at 105°C for 12 h in order to obtain a constant weight. Subsequently, 5 ml nitric acid was added to the samples so that all the samples were exposed to the acid. Subsequently, each sample was put on heater at 85ºC for 4 h until extracting the yellow smoke produced as a result of the reaction of the sample with acid. After the samples were cooled, 15 ml hydrochloric acid, 10 ml sulfuric acid, 5 ml perchloric acid, 2.5 ml trichloroacetic acid, and 1 ml oxygenated water were added to the first, second, third, fourth, and fifth samples, respectively. After passing all samples through the Whatman filter paper, they increased to a volume of 25 ml by adding double distilled water.
In the digestion method accomplished through the digestion system, 0.2 g hair color was placed on the special digestion plates. Subsequently, each sample was added with approximately 5 ml of high concentration nitric acid and 1 ml oxygenated water, according to the microwave instruction. Afterwards, the plates containing samples and acids were carefully placed in the vessels. Then, the vessels were put in the microwave, the lid was closed, and the special pressure and temperature schedule of the machine was set for the samples.
After the completion of the digestion and cooling of the device, the digested samples were extracted and added with deionized water until reaching a volume of 25 cc. Finally, it was read by the GFAAS; moreover, acetate buffer and NaHO were used to prepare samples with various pH values.
 
Preparation of samples with various pHs
In order to investigate the effect of pH applied in the sample preparation stages on the measured absorption rate by the GFAAS, nitric acid, buffer acetate (C₂H₃O₂NH₄), buffer potassium dihydrogen phosphate (KH₂PO₄), and borate buffer (Na2B4O7) were respectively used for the pH values of 2, 4, 6-8, and 10, at a volume of 1 ml. These substances were added to 5 ml of the standard levels of lead (500 ppb), cadmium (5 ppb), and chromium (200 ppb). Finally, the absorption rate was read by the GFAAS (11-12).
 
Heavy Metals Measurement Method
Initially, standard curves were drawn for the metals under investigation. The stock solutions of metal analytes were used (lead, cadmium, and chromium) for drawing standard curves (Figure1. Chart 1,2and3).

 

Figure 1) Standard curves of lead (1), chromium (2), and cadmium

After reading the standard solution absorption by the GFAAS, the original samples were subjected to the device and their absorption rates were read (µg/kg). Concentrations of the elements in the digested samples were calculated based on the standard curve equation of each metal (which was already drawn in excel). The linearity of the graph and validity of the equation were obtained by calculating R² rendering the values of 0.997, 0.991, and 0.990 for lead, cadmium, and chromium, respectively.
In order to validate and evaluate the efficiency of the digestion method and determine the reliability of the test applied for estimating the levels of heavy metals, the percent recovery of lead in hair color samples, limit of detection and limit of quantification, and intraday and interday variation coefficients were calculated. To this end, the digestion of a certain amount of metal was added to a specific sample; however, this amount was not added to the other samples. Afterward, the absorption of the two samples and the absorption of the added amount were calculated according to the following recovery formula:
R=100×(A2-A1)/ As
where R is the percent recovery, A_s is the standard adsorption, A₂ is the absorption rate of the sample containing the standard, and A₁ is the absorption of the sample without standard. In this study, to determine the percent recovery, out of the 12 hair color brands, one brand of domestic (Dopin) and one brand of foreign samples (Beauty) (showing the highest absorption rate of heavy metals) were selected to determine the percent recovery by adding 100 µl of each of 1 ppb and 5 ppb lead concentrations and brought to a volume of 10 ml (13).
After drawing the standard curve based on the standard deviation (SD) and estimated line slope, the "limit of detection" and "limit of quantitation" for both lead and chromium metals were calculated according to the following formula:
 
LOD=3 δ_b/m                      LOQ=3 LOD
 
where δ_b is the SD of six consecutive measurements of the blank solution, and m is the slope of the calculated line in the standard curve.
To evaluate the intraday variation coefficient, three lead concentrations (i.e., 50, 250, and 1250 ppb) were prepared, and their absorbance was measured six times a day by the AAS.
Furthermore, the evaluation of inter-day variation coefficient was accomplished by measuring the absorbance of three lead concentrations of 50, 250, and 1250 ppb by AAS for six consecutive days (14).
 
Statistical Analysis
Data analysis was performed in SPSS software (version 22). To compare the mean levels of lead, cadmium, and chromium between the domestic and foreign brands, independent sample t-test was employed. A p-value less than 0.05 was considered statistically significant.
 
 

Results

 
According to the results of the study, the concentration of cadmium, which was almost equal to zero, was lower than the device
limit of detection (Table 1). Moreover, the concentration of lead was reported to be higher than that of chromium in all samples (Figure2. Chart4and5).
Regarding the results presented in Table 2, there was a significant difference between the . domestic and foreign hair colors regarding the mean weight of lead and chromium concentrations. In this regard, the mean weight of lead and chromium concentrations in the domestic hair color was significantly higher
 

Table 1) Concentrations of heavy metals (µg/kg) in domestic and foreign hair color samples

Chromium	Lead	Cadmium	Manufacturer Country	Hair Color Number
6148.6	-*	-*	Foreign	Lk
-*	-*	-*	Domestic	Amousone
1243	-*	-*	Foreign	Danione
944.37	-*	-*	Foreign	Genius
35.12167	1137.5	-*	Domestic	Dopian
88.75	552.37	-*	Foreign	Biuoti
172	-*	-*	Domestic	Golbaran
-*	11.5	-*	Domestic	Albora
62.97	-*	-*	Domestic	Atousa
417.75	-*	-*	Domestic	Perestig
88	-*	-*	Domestic	Lopina
771.5	-*	-*	Foreign	Garnier

 

Figure 2) Mean concentrations of lead (4) and chromium (5) in foreign and domestic samples
 

than that in the foreign hair colors.
 
Percent recovery of lead in hair color
As mentioned earlier, percent recovery is used to measure the efficiency of the digestion method and the method used to determine the level of lead in hair color samples.
(Table 3) presents the results regarding the percent recovery of lead.
The best percent recovery is 100% but for various reasons, such as mistakes made by the researcher or the device and disturbances caused by other metals, this percentage is not usually achieved. However, the statistical results and percent recovery for the selected hair color showed that the difference of percent recovery for was not significant for lead the two added values of 1 and 5 ppb (P<0.05). Moreover, the results showed that the method used for digesting and measuring
 

Table 2) Independent sample t-test for comparing the mean logarithm of lead concentration in foreign and domestic hair colors

Heavy Metal	Manufacturer country	Descriptive statistics		Analysis variance		Compare means
		Mean	Standard Deviation	F	P-value	t	P-value
Lead	Domestic	1.49	2.83	16.963	0.001	2.122	0.048
	Foreign	0.25	1.26
chrome	Domestic	2.83	2.56	0.853	0.361	2.104	0.042
	Foreign	1.07	2.57

*Considering the difference in the variances of the two groups
 
Table 3) Mean percent recovery in the method used for extracting and measuring domestic and foreign hair color

Hair Color	Percent Recovery (Mean±SD)	Added Amount of Lead (ppb)
Dopian	98.66±1.5a	1
Dopian	97±1a	5
Biuoti	100±2a	1

 

the amount of lead in the hair color was appropriate.
 
Limit of detection, limit of quantitation, and intraday and inter-day lead variation coefficients
In order to determine the limit of detection and limit of quantitation, first, the SD and line slope were determined at a wavelength of 283.3 nm for averagely six-time test repetitions at the concentrations of 500, 1000, 1500, 2000, 2500, and 3000. After drawing a standard curve, the limit of detection and limit of quantitation for the lead were calculated as 1.5 and 4.5 ppb, respectively.
The accuracy and reproducibility of the method by using intraday and inter-day variation coefficients of lead are shown in( tables 4 and 5), respectively. According to the obtained variation coefficient, the method had a proper intraday and inter-day accuracy for

Table 4) Intraday evaluation for lead

Level (ppb)	Mean	SD	cv%
50	49.5	2	4.03
250	249	3	1.2
1250	1252	4	0.32

Table 5) Inter-day evaluation for lead

Level (ppb)	Mean	SD	cv%
50	49	2	4.07
250	251	3	1.19
1250	1252	4	0.31

 
lead measurement.
 
Effect of acid type on the absorption of heavy metals
According to (figures3. Chart 7 and 6) the absorption rate of lead when using hydrochloric acid for the digestion of the sample was higher than that for other acids. Furthermore, the absorption rate of chromium when using perchloric acid to digest the sample was higher than that of other acids.
 
Effect of Different Sample Preparation Methods on Heavy Metal Absorption
According to Figure 4, the absorption rates of lead and chromium using wet method were higher than those estimated by the dry method.
 
Effect of Sample pH Values on Absorption rate of Heavy Metal    
As depicted in (Figure5.Chart 8, 9, and 10)
 

Figure 3) Absorption rates (ppb) of lead (6) and chromium (7) using different acids in Dopin hair color
 

Figure 4) Comparison of metal absorption rate in Dopin hair color using different preparation methods
 

Figure 5) Investigation of absorption rates (ppb) of lead (8), cadmium (9), and chromium (10) at different pH values