IJERD – International Journal of Environmental and Rural Development (2017) 8-1

Research article
erd

Simplification of the Colorimetric Method to Detect Methanol
Contamination in the Cambodian Local Rice Liquor
MIZUKI MATSUKAWA*
Graduate School of Bioagricultural Science, Nagoya University, Nagoya, Japan
Email: matsukawa.mizuki@gmail.com

MITSURU HAMANO
International Center for Agricultural Education and Research, Faculty of Agriculture,
Shinshu University, Minami-minowa, JapanKASUMI ITOInternational Cooperation Center for Agricultural Education, Nagoya University, Nagoya , JapanTOSHIHARU TANAKAInternational Cooperation Center for Agricultural Education, Nagoya University, Nagoya , Japan
Received 16 January 2016

Accepted 26 June 2017(*Corresponding Author)

Abstract Methanol, an alcohol, is known to be hazardous for human consumption. Methanol
contamination in traditional rice liquor caused many deaths in local areas in Cambodia.
Contamination happened in every step of liquor manufacturing, distribution, and consumption.
To avoid this problem, monitoring the quality of alcohol is important. However, only a few
government institutes in the capital can detect the methanol contamination at an institutional
level by colorimetric methods. To detect methanol contamination easily at the local level, asimplified method is urgently required. We tested the original colorimetric methods todetermine the influence of the amount of chemical solutions, the time and the alcohol
percentage to the color change. Further we checked the shelf life of the chemical solutions. The
results showed that methanol was detectable at one-twenty of original volume after treatment 25 hours, and the alcohol percentage was not influence of the color changes. In addition, wetested 21 liquor samples collected from markets in Phnom Penh and 6 provinces with thesimplified method, resulting that methanol was not detected in all samples.
Keywords local liquor, methanol, simplified methods

INTRODUCTIONMethanol, an alcohol, is known to be hazardous for human consumption. In the worst-case scenario,
symptoms of methanol poisoning caused by inhalation or intake are blindness and death (Bennet et al.
1953). Recently, newspapers have reported that the contamination of methanol in traditional rice liquor
caused many deaths in local areas of Cambodia (Gee & Martin 2012, The Cambodia Daily 2011).
People bought cheap industrial alcohol at a high concentration, and mixed it as a loading fluid in every
step of liquor manufacturing, distribution, and consumption.
Usually, when the equipment is available, the detection of methanol contamination is performed
by gas chromatography, which is a quick and accurate method. However, due to high cost and shortageof human resources, this might be difficult to perform in developing countries.
In Cambodia, the Industrial Laboratory Center of Cambodia and CAMCONTROL in Phnom Penh
have the resources to monitor the quality of alcohol at an institutional level using colorimetric methods.
However, government agencies for the control of the quality of alcohol have not been established inⒸ ISERD150

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other provinces. Therefore, the establishment of a simple method to detect methanol contamination
based on the existing protocol is required promptly to decrease the number of fatal accidents occurringwith traditional rice liquor in Cambodia or other developing countries.
OBJECTIVESIn this study, we improved some aspects of the conventional colorimetric method for detecting
methanol by visual analysis. In order to simplify the colorimetric method, we aimed to modify thefollowing 4 aspects: (1) detection sensitivity depending on reduced amount of chemical solutions, (2)optimum time to check the solution color after the final treatment, (3) detection sensitivity dependingon the alcohol percentage, and (4) retention period of the chemical solutions. In addition, we conducted(5) methanol detection in rice liquors available in the local markets from 6 provinces.
METHODOLOGY
Original Method
We followed the colorimetric method reported by the Japan National Tax Agency in 1961(NationalTax Agency of Japan 2007). A brief description of the method is reported below.
Preparation of chemical solutions: Solution A (500 mL): 15 g of potassium permanganate (VII)(KMnO4) and 75 mL of phosphoric acid (H3PO4) were dissolved in distilled water and up to 500 mL,Solution B (500 mL): 25 g of oxalic acid dihydrate [(COOH)2・2H2O] was dissolved in 500 mL 50%(v/v) sulfuric acid (H2SO4), Solution C (500 mL): 0.5 g of basic fuchsin was dissolved in about 300 mLof distilled water boiled at 100°C, and 5 g of sodium sulfite (Na2SO3) was dissolved in about 50 mL ofdistilled water. Fuchsin solution was mixed with Na2SO3 solution and 5 mL of hydrochloric acid (HCl)was added. Then, the final solution was diluted to 500 mL with distilled water. Finally, 40 mL of 10N
H2SO4 was added, and the solution was kept at room temperature for more than 5 h. The color often
became light brown after adding HCl. Although it should be used after the color of fuchsin disappear,
it could be a pale yellow color solution. The all chemicals were purchased from Wako Pure ChemicalIndustries.
Preparation of standard solutions: The standard solutions were prepared with 99.5% ethanol andmethanol (Wako Pure Chemical Industries, Ltd). Ethanol was adjusted to 40% (v/v) with distilledwater, and 0, 5, and 15µL methanol was added into 10 mL of 40% ethanol to make the 0, 0.05, and0.15% (v/v) methanol artificial contaminated solutions since the Cambodian government set 0.15% is
the highest standard level of methanol contamination in the original liquor. Finally these mixtures were8 times diluted to prepare the 5% alcohol percentage for testing.
Test procedures according to original method: The sample solution (5 mL) was mixed in a test tube
with 2 mL of solution A and left for 10 min. Then, 2 mL of solution B and 5 mL of solution C wereadded in this order and mixed well. After 30 min, the color of the solution was checked and theabsorbance was determined at 590 nm with a spectrophotometer (U-2000A, HITACHI) (Hayashibe1955) using low volume cell. Checking the absorption wavelength of standard solution determined thepeak at 580-600 nm, hence we adopted 590 nm as the wavelength for the measurement. The colors ofmixture turned from clear and colorless (0%), weak purple (0.15%) to purple (over 0.8%) according tothe trial experiment.
Simplification of the Original MethodⒸ ISERD151

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The 40% ethanol with methanol 0, 0.05 and 0.15% (v/v) was diluted into 5% as the standard solutions.
All steps were conducted under the room temperature (about 22-25°C).
Dose reduction of the chemicals: Various total amounts of standard and chemical solutions (i.e., 1,1/2, 1/5, 1/10 and 1/20 of the amount indicated in the original protocol) were used to detect themethanol contamination in standard solutions. The color of the solution was assessed by visual analysis
and spectrophotometer (590 nm) in following analyses.
Optimum time to check the color of the solution: The change of color was assessed 0.5, 1, 2, 3, 5,
and 24 h after adding the final solution C. The amounts of solution were as follows: standard 0.25 mL,
solution A 0.1 mL, solution B 0.1 mL, and solution C 0.25 mL.
Difference in the sensitivity depending on the percentage of alcohol in the sample solution: We
compared the sensitivity of the colorimetric method between ethanol standard solutions (5%) andoriginal solutions (40%) respectively. Further, rice liquor samples produced in Cambodia (Sraa Takeo
40% alcohol/volume, Royal University of Agriculture, Phnom Penh, Cambodia) were used; original
percentage of the liquors (40%) with 0, 0.05 and 0.15% methanol artificial contamination, and its 5%
diluted solutions. The amounts of solution were as follows: ethanol/liquor solutions (40% or 5%) 0.25mL, solution A 0.1 mL, solution B 0.1 mL, solution C 0.25 mL. The color was checked 3 h aftertreatment.
Expiration period of chemical solutions: Test solutions were kept for 2 and 4 weeks at room
temperature (about 22–25°C) and, then, were tested in comparison with fresh solutions. The
combination of the solutions is shown in Table 1; N: fresh solution and O: 2 or 4 weeks old solutions.
The amounts of the solutions were as follows: standard solutions 0.25 mL, solution A 0.1 mL, solution
B 0.1 mL, and solution C 0.25 mL. The color was checked 3 h after treatment.
Table 1 The combinations between fresh and old solutions
TestsolutionABC

1NNN

2ONN

3NO
N

Combinations*45NO
NOO
N

6ONO

7NOO

8OOO

* N: fresh solutions, O: 2 or 4 weeks old solutions

Detection of methanol from the local rice liquors: Rice liquors collected from the local market in
Phnom Penh and 6 provinces (Battambong, Kompong Chhunang, Prey Veng, Pursat, Svay Rieng,Takeo) were used as liquor samples; 3 samples/province and 21 samples in total. Rice liquors wereclear and colorless and the original liquors were diluted into 5% for testing. The amounts of thesolutions were as follows: liquor 0.25 mL, solution A 0.1 mL, solution B 0.1 mL, and solution C 0.25
mL. The color was checked 3 h after treatment.
Data analysis: The data was analyzed using with Excel statistical analysis add-in software (Excelstatistic 2012; Social Survey Research Information Co., Ltd., Tokyo, Japan).
RESULTS AND DISCUSSION
The methanol percentage in the all figures indicated the methanol percentage in the originalconcentration (0, 0.05 and 0.15%), and the absorbance values of solutions (original/5% diluted) wereshown without change.
Scale-down of the amount of chemical solutions: The reduction of the waste chemical solutions isrequired because the disposal of the toxic waste in Cambodia is not yet well-implemented and heavy
metal such as KMnO4 pollutes the environment when discarded without treatment. We could
recognized the difference between 0 and 0.15% methanol contamination by eyes in every total amount,Ⓒ ISERD152

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and the absorbance among 0, 0.05, and 0. 15% methanol were significantly different in 1/20 of theamount in the original protocol (Fig. 1). It shows that methanol contaminations in the smallest amountof chemical solutions (ethanol standard: 0.25 mL, A: 0.1 mL, B: 0.1 mL and C: 0.25 mL) could be
detected as same as original amount.
cbaFig. 1 Scale-down of the amount of chemical solutions
Significant differences among each methanol concentration of 1/20 of the initial solution weredetermined by one way-ANOVA. The same letters (a-c) mean no significant difference (p<0.01).

Optimum time for detecting the color of the solution: The optimum time for comparing the color byvisual judgment was determined. 0.15% methanol, the highest allowed level of methanol
contamination decided by the Cambodian government, was visually detected 0.5 h after adding thefinal solution. Two hours later, the differences of the color between 0% and 0.15% methanol could beobserved easier, while no difference could be detected visually between 0% and 0.05% samples.
However, after 24 h, although the difference of absorbance was detected by spectrophotometer, nodiscernible difference could be observed. Therefore, we can check the color of the solutions 2–5 h aftertreatment (Fig. 2).

Fig. 2 Optimum time for detecting the color of the solution
Detection sensitivity for methanol contamination depending on the alcohol percentage: The
detection sensitivity of the 40% ethanol/rice liquor were compared with 5% diluted ethanol/ rice liquor
to know whether methanol contamination can be detected in the original percentage. We could notrecognize visually the difference between 0 and 0.05% methanol in both samples, however between
0% and 0.15%, it was detectable the difference by naked eyes though the absorbance was lower inoriginal percentage than in 5% diluted one of both ethanol and liquor solutions (Fig. 3). To detect theaccurate amount of methanol in alcoholic beverage, though gas chromatography is so convenienceⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2017) 8-1(Wang et al. 2004), the method shown in this study makes us possible to quick detection and visual
discrimination if concentration of methanol included in the beverage is higher than specified safetylevel without using special equipments.

Fig. 3 Difference of detection sensitivity for methanol contamination
depending on the alcohol percentage
Concentration-dependent change of solution color of (a) ethanol standard (5%) and (b) rice liquor (40%) with 0, 0.05, and
0.15% methanol contaminations from left to right, (c) concentration-dependent change of absorbance among 0, 0.05, and
0.15% methanol contaminations in 5% and 40% ethanol/rice liquor. The same letters (a-c) mean no significant difference(p<0.01) of the 40% rice liquor among each methanol concentrations as determined by one-way ANOVA.

Expiration period of the chemical solutions: To save time for the preparation of the chemicals, theretention period of chemical solutions was determined. The combination of fresh chemicals with oldsolutions was tested at 2 and 4 weeks after preparation. The detection sensitivity of the chemicalsolutions was not changed after 2 weeks. However, after 4 weeks, a difference could be observed andthe detection sensitivity became lower in conditions 2, 4, 5, 6, 7, and 8, which include old A and Csolutions. On the other hand, in condition 3, which includes old B solution, the color was the same asthat of the normal fresh condition (Table 1, Fig. 4).

Fig. 4 Expiration period of each chemical solution affecting detection sensitivity
Dot, diagonal line, and black bars represent the absorbance of the 5% ethanol solution containing 0, 0.05, and 0.15%
methanol, respectively. Conditions 1-8 show different combinations with fresh and old chemicals of A, B, and C (Table 1).
Significant differences among conditions in (a) fresh and 2-week-old solutions, (b) fresh and 4-week-old solutions werecalculated with one-way ANOVA respectively. The same letters mean no significant difference (p<0.05): 0.05% (a-e) and
0.15% methanol (s-v) among condition 1-8.

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Detection of methanol from the local rice liquors: The 21 local rice liquor samples were tested bythe simplified method. No methanol contamination was detected from those samples.
Table 2 Detection of methanol contamination from local rice liquors
Province
Battambong
PursatKompong Chhunang
Phnom Penh
Prey Veng
Svay Rieng
TakeoTotal

Number ofsample
333333321

Methanolcontamination
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected
Not detected

CONCLUSION
Based on our observations, we conclude that: (1) the amount of solutions can be reduced to a total
volume of 0.7 mL (standard 0.25 mL, solution A 0.1 mL, solution B 0.1 mL, and solution C 0.25 mL);(2) the difference of color can be detected easily 2-5 h after the final treatment; (3) Moreover, themethanol contamination was detected without dilutions, and all the degrees of methanol contamination
could be identified by comparison with 0% methanol standard solution though the detection ability was
higher in 5% diluted solutions; (4) The degradation of the solutions increased with time, especially forsolution C; therefore, the results indicated that solutions A and C could be used for 2 weeks, andsolution B for 4 weeks. It is preferable to prepare fresh solutions within 2 weeks considering that thevolatile substances in the solutions are hazardous; (5) Furthermore, this method can be used for rice
liquor from local markets. Our results suggested that the simplified methods could identify themethanol contamination over the highest standard level by visual though it couldn’t be measured
accurate percentage of methanol contamination. Once the contamination is detected, the rice liquor isrequired to be analyzed in detail by laboratory experiments.
ACKNOWLEDGEMENTS
We thank the staff of “JICA Grass-Root Project on Development of Agro-processing Technology
through Rehabilitation of Traditional Industry in Cambodia”.
REFERENCES
Bennett, I.L. et al. 1953 Acute methyl alcohol poisoning, A review based on experiences in an outbreak of323cases. Medicine, 32, 431-63.
Gee, P. and Martin, E. 2012. Toxic cocktail, Methanol poisoning in a tourist to Indonesia. Emergency Medicine
Australasia, 24, 451-453.
Hayashibe, R.1955. Quantitative determination of methylalcohol (First report). Study of condition of photometric analysis
according US methods. Nihon jozo kyokai (Journal of the brewing Society of Japan), 50, 59-61 (in Japanese).
Wang, M.L. et al. 2004. A rapid and accurate method for determination of methanol in alcoholic beverage bydirect injection capillary gas chromatography. Journal of Food Composition and Analysis, 17, 187-196.
National Tax Agency of Japan. 2007. Kokuzeicho shotei bunsekiho (National Tax Administration Agency
prescribed analytical methods), Tokyo, Japan, 36 (in Japanese).
The Cambodia Daily. http://www.cambodiadaily.com/archive/rice-wine-poisoning-leaves-a-village-reeling-2885/Ⓒ ISERD155