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Running head: BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Bioluminescence Method as a Screening Test of Mercury Content in Whitening Products

Andoy, Kris Kate C. De Luna, Joseph G. Sambrano, Allyssa Loraine C.

Applied Science - Team

Pacita Complex National High School

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Title: Bioluminescence Method as a Screening Test of Mercury Content of Whitening Products

Proponents: Andoy, Kris Kate C. De Luna, Joseph G. Sambrano, Allyssa Loraine C.

Research Adviser: Clarence V. Bellen

School: Pacita Complex National High School

Over the years, there had been a growing concern in mercury poisoning in public which can cause potentially deadly health problems. Its compounds are highly toxic. Exposure to mercury can cause gastrointestinal problems and kidney damage. Last June 2017, the Department of Environment and Natural Resources (DENR) and the Department of Health (DOH) stated that there are approximately 10,000 residents in two barangays in Puerto Princesa, Palawan, Philippines are in danger of being gravely affected by mercury poisoning. According to Simoune A. (2016), the danger of mercury contamination is not only on the people who use the product but also to their families. The mercury vapours released from these products can be inhaled by children, especially pregnant woman and can affect to the development of brains and nervous system of the infants. Mercury is also found on cosmetics mostly in whitening products. Sec. 700.13, “Use of Mercury Compounds in Cosmetics Including Use as Skin Bleaching Agents in Cosmetic Preparations also Regarded as Drugs” of Food and Drug Administration (FDA) stated

1

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT that cosmetics should have contain mercury at most of 1 part per million (0.0001 %) and if the cosmetics is intended for use only in the area of the eye it contains no more than 65 parts per million (0.0065 %). Aliivibrio fischeri is known as a bioluminescent bacteria living in an aquatic environment. This bacterium is not pathogenic to humans. According to Quinto E. (2012), bioluminescence can be a new method in determining the presence of water toxicants. The light given by living organisms produced from their metabolic activity, often called “Living Light” or “Cold Light”, bioluminescence can be used to tell that a toxicant is present in the water sample precluding its domestic use though cannot identify the toxicant/s present in a water sample. Bioluminescence- based Technology is a “Green Technology” which is a “Less is More” alternative sustainable process that reduces toxicity, carbon footprints, energy consumption and greenhouse gas emissions. The researchers aim to prove if the bioluminescence method can determine the presence of mercury on different whitening products. Background of the Study Whitening cosmetic is one of the widely used products all over the world. It is known to whiten skin because of its different chemical composition like mercury, but too much can lead to a serious damage. According to World Health Organization (WHO), long exposure to mercury may impact on human health such as on the nervous system, digestive and immune systems, and on lungs, kidneys and eyes. It is also stated that there is no safe level of mercury.

2

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Mercury can also be found on our environment. World Health Organization (WHO) stated that it has been estimated that 17 out of 1000 children shows a cognitive impact on their health by consuming fish with mercury. Mercury on the environment was mainly caused by different human activities like coal-fired power stations, residential heating systems, and waste incinerators and as a result of mining for mercury, gold and other metals. Once in the environment, elemental mercury is naturally transformed into methylmercury that bio accumulates in fish and shellfish. To prevent mercury contamination, the government assigns the Food and Drug Administration (FDA) to check every cosmetic for its harmful effects on human before it would be out in the market. Government also implements some laws regarding sanitation. Presidential Decree no. 856, Chapter III, Section 32, G. stated that all fish caught with high mercury content should be condemned and not be allowed for public consumption. But still, there are cases of mercury contamination regarding the use of cosmetics especially whitening products. So the researchers intend to determine if the bioluminescence method can determine the presence of mercury on different whitening products. Bioluminescence in the Sea by Case J., Haddock S., and Moline M. (2010) Bioluminescence is the emission of light by an organism as a result of a natural chemical reaction such remarkable diversity of aquatic animals and microbes are able to produce their own light. It has been found across a broad range of the major groups of organisms from bacteria and protists to squid and fishes, with numerous phyla in between. Typically, it is produced by the oxidation of a light-emitting molecule.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Bioluminescent bacteria are commonly found in the ocean, especially in temperate to warmer waters. Bacteria are not luminous until they have reached sufficiently high concentrations to initiate quorum sensing, and once induced; they glow continuously in the presence oxygen rather than producing discrete flashes. Gram-negative y. proteobacteria specifically from so-called eubacteria, among the prokaryotes, is the only one that has the capability of light production. The genus of Vibrio is the best studied symbiotic bacteria, although the genus of Shewanella also includes a bioluminescent species. Bioluminescence is an extremely effective way for invertebrates to communicate to organisms much larger and potentially far away and this may help explains the prevalence. Recent Developments in the Speciation and Determination of Mercury (Hg) Using Various Analytical Techniques by Suvarapu L. and Sungok B. (2015) Mercury is also known as quick silver. It is only metal that exist in liquid form at room temperature. The toxicity and bioaccumulation nature of mercury has prompted extensive studies to determine the concentrations of mercury species in different environmental and biological samples. The research concluded that cold-vapor atomic absorption spectrometry (CV-AAS) and atomic absorption spectroflourometry (CV-AFS) for the speciation and determination studies of mercury in various environmental samples.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Evidence of Bacterial Bioluminescence in a Philippine Squid and Octopus Hosts by Naguit M., Abisado R., Plata K., et.al (2014) Bioluminescence has been extensively studied typically in squid and fishes hosts and in other aquatic organisms. The different organisms (Squid and Octopus) were purchased from the seafood market Cubao, Quezon City, Metro Manila Philippines. Luminescence swabs was taken inside of the organism specifically in the head, body, and eye part and streaked on luminescence agar. The result shows that between the organisms, octopus samples were found to harbour bioluminescent bacteria. Also in squid they also harbour bioluminescent on the head area. Cyanide and Mercury Level Determination in Small Scale Mining Areas in the Philippines by Lu J. (2014) Mercury is one of the chemicals extensively used in mining to extract microscopic gold particles. It was estimated that there were about 1.6 tons of Mercury released for every ton of gold produced in the Philippines. To analyse the cyanide and mercury residue in the water samples, they used Atomic Absorption Spectroscopy. The study has shown the possible association between the prevalence of mercury and cyanide readings with current and past wining activities in the area. Statement of the Problem This study was undertaken to assess the chemical toxicity of mercury using the bioluminescence method and its relation to the chemical present in whitening products. Specifically, its sought achieving the following goals;

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT 1. Determine the visible spectra of bioluminescence in whitening products using Spectrometry. 2. Determine the mercury content of whitening product samples using ICP-OES. 3. Determine the visible spectra of bioluminescence after 50 minutes exposure in whitening product samples using spectrometry. 4. Measure the intensity measurement of bioluminescence in whitening product samples using Image processing software.

5. Determine the significant difference in the intensity of light produced by bioluminescent bacteria between the whitening product samples. 6. Determine the significant association between the intensity of light produced by bioluminescent bacteria to the mercury content of whitening product samples. Hypotheses The researchers’ were guided by the hypotheses stated in null and alternative form. Set A Ho – There is significant difference in the intensity of light of bioluminescence after 50 minutes between whitening products. Ha – There is a significant difference in the intensity of light of bioluminescence after 50 minutes between whitening products.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Set B Ho –There is no significant association between the intensity of light produced by the bioluminescence and the mercury content of whitening product samples. Ha – There is a significant association between the intensity of light produced by the bioluminescence and the mercury content of whitening product samples. Significance of the Study The findings of this study will provide information and data significant to several groups. Microbiologist. This study could give them new information on how the rapid bioluminescence reacts on the mercury content of a product. This could also lead to a new function of bioluminescent bacteria (Aliivibrio fischeri). Manufacturers and Consumers. This study would serve as an eye opener to those manufacturers about the effects of high mercury content to humans. Community. The study would help the people in the community to know if their cosmetics contain the presence of mercury. Government. The government can easily screen the presence of mercury on the whitening products that will be out from the market. Future Researchers. The future researchers can create a device that would be available in the market to easily identify the presence and the amount of mercury.

7

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Scope and Limitations The study is only limited on determining the presence of mercury in whitening products using the bioluminescence method. It only involves the fischeri specie of the Aliivibrio genus and does not include other species. Each set-up of the experiment will show the presence of mercury on whitening products by means of the intensity of the light and visible spectra produced by the bioluminescent Aliivibrio fischeri. To determine the mercury content of whitening product samples, the samples will be brought in Adamson University Technological and Research Developmental Center (AUTRDC) and will undergo Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). The intensity of the light will be measured using an image processing program while the visible spectra will be measured using a spectrometer. The image processing program that will be used is ImageJ that is powered by Java. USB-650 Red Tide Spectrometer that have a wavelength range of 380 nanometer (nm) to 1000nm and sensitivity of 400 photons/ 400 nm will be used. II. Material and Methods This chapter contains the Research Design, Sampling Technique, Research Instruments, Procedure and Experimental Design that will be used in conducting the study. Research Design The research study is experimental that consist of one treatment arrange in Single Group Design. The treatment is classified as the whitening products labelled as X, Y and

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Z. The treatment will be placed in different test tubes containing the bioluminescent Aliivibrio fischeri. Sampling Technique The researchers employ Purposive Sampling in the acquisition of Aliivibrio fischeri and the whitening products. These were chosen because of its purpose in conducting the experiment. The Aliivibrio fischeri strain will be isolated from squid’s ink while the whitening products will be bought from different shopping centers. Research Materials A. 30 pcs. of Disposable Inoculating loop B. 20 pcs. of Disposable petri dish C. 9 pcs. Test Tubes D. 2 Erlenmeyer Flask E. Stirring Rod F. 5 pcs. of Spatula G. 1 pc. of Funnel H. Thermometer I. 10 pcs. of 50 mL Beaker J. 1 pc. of 1000 mL Beaker K. 20 g of Tryptone L. 18g of Nutrient Agar M. ¼ kg of Fresh Squid N. Hot plate

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT O. Weighing Scale P. Whitening Products (Brand X, Y, and Z) Flowchart of the Research Procedure

Preparation of Whitening Products for Spectrometry Preparation of Luminescent Bacteria (Aliivibrio fischeri) Bioluminescence Phenomenon

Preparation of Whitening Product Samples for Rapid Bioluminescence Method

Observation and Interpretation of Results

Research Procedure 1.

Preparation of Whitening Products for Spectrometry A variety of whitening products will be obtained randomly from a shopping center

from different manufacturing companies and will be labelled as brand X, Y, and Z. The whitening product samples will undergo serial dilution to get the concentration of 0.01%. The visible spectra of whitening product samples with 0.01 v/v concentration will be get using USB-650 Red Tide Spectrometer.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT 2.

Preparation of Luminescent Bacteria (Aliivibrio fischeri) The luminescent bacteria (Aliivibrio fischeri) will be isolated from the squid. The

squid will be bought at the San Pedro Public Market; Laguna, Philippines early in the morning from 5:00 a.m. to 7:00 a.m. to obtain freshly caught samples. The ink of the squid will be streaked by multiple times on a Tryptone Yeast Extract Glycerol Salt Water Plate and incubated at room temperature for 10 hours. After all the components were dissolved and the pH has been adjusted to 6 using 1 M of hydrochloric acid or 1 M of Sodium Hydroxide Solution, culture media was sterilized on a hot plate at 110°C. 3.

Bioluminescence Phenomenon A brightly luminous colony from the species will be chosen from the plate, fished

out using inoculating loop and will be inoculated into freshly prepared artificial sea water, 250 ml in Erlenmeyer flask. A 4ml of bioluminescent bacteria will be placed in different test tube containing 5mL artificial sea water. 4.

Preparation of Whitening Product Samples for Rapid Bioluminescence Method 4 drops of each whitening product samples will be placed in each test tubes,

labelled with Brands X, Y and Z. Every 5 minutes the test tubes will be photographed using Canon 700D in manual setting with a 30 seconds exposure. 5. Observation and Interpretation of Results The intensity of the light will be measured using an image processing program while the visible spectra will be measured using a spectrometer.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Research Instrument Table 1. The Amount of Mercury Content of Whitening Product Samples (Brand X, Y, and Z) The Amount of Mercury Content of Whitening Product Samples by ICP OES Brands

Amount (percentage %)

Table 1 will show the amount of mercury present in each whitening product samples using the Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Table 2. Intensity of Light produced by the Bioluminescence in Whitening Product Samples (Brand X, Y, and Z) Intensity of Light produced by the Bioluminescence in Whitening Product Samples Time (Minutes) 0 5 10 15 20

X

Y

Z

12

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT 25 30 35 40 45 50 Mean

This table shows the intensity of light by Bioluminescent Aliivibrio fischeri in minutes. The means will be used for comparison of intensity of light of bioluminescence on whitening product samples to the amount of mercury content. Table 3. Rate of Decay in Intensity

RATE OF DECAY IN INTENSITY X Y Z

Table 3 will show the rate of decay or how fast the intensity of light produced by bioluminescent bacteria decrease. This will also show the effect of whitening product samples on the intensity of light by bioluminescence.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Risk and Safety The researchers will conduct a study about the bioluminescence method as a screening test of mercury on whitening products with the following risk and safety: 1. Safety measures are needed on the acquisition of whitening products that contain mercury. 2. Use of laboratory gown and hand gloves while handling the bacterial strain. 3. Sterilize equipment and materials before and after using it. 4. Label every materials used clearly. 5. Autoclave or disinfect all waste materials and disposed it properly.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Bibliography Case J., Haddock S., and Moline M.(2010) Bioluminescence in the Sea. Retrieved September 2, 2017, from https://www.researchgate.net/publication/49667852 CrawLaw.(n.d.). Philippine Environmental Laws – Chan Robles Virtual Law Library. Retrieved September 2, 2017, from http://www.chanrobles.com/presidentialdecreeno856.htm#.WaoyrYVOLIU

Formoso, C. (2017). Poisoned lake, quaysides threaten residents in 2 Puerto Princesa villages. Philippine News Agency. Retrieved June 7, 2017, from http://www.pna.gov.ph/articles/994121 Lu J. (2014). Cyanide and Mercury Level Determination in Small Scale Mining Areas in the Philippines. Retrieved September 2, 2017, from www.tsijournals.com

Naguit M., Abisado R., Plata K., et.al (2014).Evidence of Bacterial Bioluminescence in a Philippine Squid and Octopus Hosts. Retrieved September 2, 2017, from http://www.bioflux.com.ro/aac

Quinto, E. (2013) Simple water toxicity test using Photobacterium leiognathi Retrieved August 25, 2017, from http://web.a.ebscohost.com/ehost/detail/detail?sid=6e0c495685104974920181828c 569ac9%40sessionmgr4005&vid=0&hid=4209&bdata=JnNpdGU9ZWhvc3QtbGl2 ZQ%3d%3d

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT Sasaki, S., Okamoto, T. and Fujii, T. (2009) Bioluminescence intensity difference observed in luminous bacteria groups with different motility. Lett Appl Microbiol 48, 313–317. Retrieved: September 10, 2017 From: http:// onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2008.02525.x/full

Simoune A. (2016). Mercury Poisoning Linked to Skin Products. Retrieved August 25, 2017, from https://www.fda.gov/ForConsumers/ConsumerUpdates/ucm294849.htm Suvarapu L. and Sungok B.(2015). Recent Developments in the Speciation and Determination of Mercury (Hg) Using Various Analytical Techniques. Retrieved September 4, 2017, from https://www.hindawi.com/journals/jamc/372459/ U.S. Food and Drug Administration.(n.d.). CFR - Code of Federal Regulations Title 21. Retrieved August 25, 2017, from https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=700.1 3 World Health Organization. (n.d.). International Programme on Chemical Safety, Mercury. Retrieved September 2, 2017, from http://www.who.int/ipcs/assessment/public_health/mercury/en/

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Running Head: BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Bioluminescence Method as a Screening Test of Mercury Content in Whitening Products

Andoy, Kris Kate C. De Luna, Joseph G. Sambrano, Allyssa Loraine C.

Applied Science - Team

Pacita Complex National High School

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Abstract A variety of whitening products contain mercury which imposes a health concern. This study was undertaken to assess the chemical toxicity of mercury using the bioluminescence method and its relation to the chemical present in whitening products. Different whitening products were gathered and labelled as Brands X, Y, and Z. Aliivibrio fischeri were isolated from the squid’s ink and were streaked multiple times at Tryptone Yeast Extract Glycerol Salt Water Plate and was incubated at 24 oC for 10 hours. A brightly luminous colony was harvested and inoculated into freshly prepared artificial sea water. 4 drops of each whitening product in 0.01 v/v concentration was placed in each test tube. Result of ICP – OES showed that brand X has the highest mercury content with 0.589 % while brand Z have the lowest mercury content with 0.182%. Spectrometry proved that brand Y has the highest related intensity ranging from 0% to 0.42% among the whitening product samples. The relative intensity of bioluminescence in whitening products decreases by 0.13% to 0.01%, 0.22% to 0.1 %, and 0.16 % to 0.1% respectively. Brand X is the fastest while brand Z is the slowest. The computed F is 7.232 which exceeds Fo.o1 = 5.3905 that means the intensity of light of bioluminescence differs significantly across whitening products with different mercury content. The computed R is -0.991 which indicates very high correlation and highly significant (p< 0.05). As the mercury content of whitening product increases the intensity of light of the bioluminescence decreases and vice versa. Keywords: Bioluminescence, Aliivibrio fischeri, Mercury, Spectrometry, Inductively Coupled Plasma – Optical Emission Spectrometry (ICP=OES)

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Table of Contents Page no. I.

Introduction

1

a.) Statement of the Problem

3

b.) Objectives

3

c.) Hypotheses

4

d.) Significance of the Study

5

e.) Scope and Limitations

5

II. Materials and Methods

6

a.) Flowchart of the Methodology

8

b.) Research Instrument

8

c.) Risk and Safety

10

d.) Data Analysis

11

III. Results and Discussion

12

IV. Conclusion and Recommendation

27

a.) Conclusion

27

b.) Recommendation

28

V. Bibliography VI. Acknowledgement VII. Dedication VIII. Appendices

1

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Bioluminescence Method as a Screening Test of Mercury Content of Whitening Products Over the years, there had been a growing concern in mercury poisoning in public which can cause potentially deadly health problems. Its compounds are highly toxic. Exposure to mercury can cause gastrointestinal problems and kidney damage. According to World Health Organization (WHO), long exposure to mercury may impact on human health such as on the nervous system, digestive and immune systems, and on lungs, kidneys and eyes. It is also stated that there is no safe level of mercury. The danger of mercury contamination is not only on the people who use the product but also to their families. The mercury vapours released from these products can be inhaled by children, especially pregnant woman and can affect to the development of brains and nervous system of the infants. (Simoune 2016) Mercury can also be found on our environment. Last June 2017, the Department of Environment and Natural Resources (DENR) and the Department of Health (DOH) stated that there are approximately 10,000 residents in two barangays in Puerto

,

Palawan, Philippines are in danger of being gravely affected by mercury poisoningg. It has been estimated that 17 out of 1000 children shows a cognitive impact on their health by consuming fish with mercury. Mercury on the environment is mainly caused by different human activities like coal-fired power stations, residential heating systems, and waste incinerators and as a result of mining for mercury, gold and other metals. Once in the environment, elemental mercury is naturally transformed into methylmercury that bio accumulates in fish and shellfish. Whitening cosmetic is one of the widely used products all over the world. It is known to whiten skin because of its different chemical composition like mercury, but too much can lead to a serious damage. The Sec. 700.13 entitled “Use of Mercury Compounds in Cosmetics Including Use as Skin Bleaching

2

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Agents in Cosmetic Preparations also Regarded as Drugs” of Food and Drug Administration (FDA) stated that cosmetics should have contain mercury at most of 1 part per million (0.0001 %) and if the cosmetics is intended for use only in the area of the eye it contains no more than 65 parts per million (0.0065 %). To prevent mercury contamination, the government assigns the Food and Drug Administration (FDA) to check every cosmetic for its harmful effects on human before it would be out in the market. Government also implements some laws regarding sanitation. Presidential Decree no. 856, Chapter III, Section 32, G. stated that all fish caught with high mercury content should be condemned and not be allowed for public consumption. But still, there are cases of mercury contamination regarding the use of cosmetics especially whitening products. Aliivibrio

fischeri

is

known

as

a

bioluminescent

bacteria

producing

bioluminescence living in an aquatic environment. This bacterium is not pathogenic to humans. Bioluminescence is the emission of light by an organism as a result of a natural chemical reaction, bacteria are not luminous until they have reached sufficiently high concentrations to initiate quorum sensing, and once induced, it will glow continuously in the presence oxygen rather than producing discrete flashes (Case et.al, 2010) Bioluminescence can be a new method in determining the presence of water toxicants. The light given by living organisms produced from their metabolic activity, often called “Living Light” or “Cold Light”, bioluminescence can be used to tell that a toxicant is present in the water sample precluding its domestic use though cannot identify the toxicant/s present in a water sample. Bioluminescence- based Technology is a

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

“Green Technology” which is a “Less is more” alternative sustainable process that reduces toxicity, carbon footprints, energy consumption and greenhouse gas emissions. (Quinto 2012) Mercury contamination is of great concern to the public, as it has been wide threat. A variety of whitening products imposes a major health concern of this chemical. This study focused in assessing the mercury content using rapid bioluminescence method. It aimed to measure the extent to which light production by the bacteria is inhibited in relation to the amount of mercury contained on each whitening product samples. Statement of the Problem This study was undertaken to assess the chemical toxicity of mercury using the bioluminescence method and its relation to the chemical present in whitening products. Objectives This research sought achieving the following goals: 1. Determine the visible spectra of bioluminescence in whitening products using Spectrometry. 2. Determine the mercury content of whitening product samples using ICP – OES. 3. Determine the visible spectra of bioluminescence after 50 minutes exposure in whitening product samples using spectrometry. 4. Measure the intensity measurement of bioluminescence in whitening product samples using Image processing software.

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

5. Determine the significant difference in the intensity of light produced by bioluminescent bacteria between the whitening product samples. 6. Determine the significant association between the intensity of light produced by bioluminescent bacteria to the mercury content of whitening product samples. Hypotheses The researchers’ were guided by the hypotheses stated in null and alternative form. SET A Ho – There is no significant difference in the intensity of light produced by bioluminescent bacteria between the whitening product samples. Ha – There is a significant difference in the intensity of light produced by bioluminescent bacteria between the whitening product samples. SET B Ho – There is no significant association between the intensity of light produced by the bioluminescence and the mercury content of whitening product samples. Ha – There is a significant association between the intensity of light produced by the bioluminescence and the mercury content of whitening product samples.

4

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Significance of the Study The findings of this study will provide information and data significant to several groups. Microbiologist. This study could give them new information on how the rapid bioluminescence reacts on the mercury content of a product. This could also lead to a new function of bioluminescent bacteria (Aliivibrio fischeri). Manufacturers and Consumers. This study would serve as an eye opener to consumers and manufacturers about the effects of high mercury content to humans. Community. The study would help the people in the community to know if their cosmetics contain the presence of mercury. Government. The government can easily screen the presence of mercury on the whitening products that will be out from the market. Future Researchers. The future researchers can create a device that would be available in the market to easily identify the presence and the amount of mercury. Scope and Limitations The study is only limited on determining the presence of mercury in whitening products using the bioluminescence method. It only involves the fischeri specie of the Aliivibrio genus and does not include other species. Each set-up of the experiment will show the presence of mercury on whitening products by means of the intensity of the light and visible spectra produced by the bioluminescent Aliivibrio fischeri. To determine the mercury content of whitening product samples, the samples will be brought on

5

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Adamson University Technological and Research Developmental Center (AUTRDC) and will undergo Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES).The intensity of the light will be measured using an image processing program while the visible spectra will be measured using a spectrometer. The image processing program that will be used is ImageJ that is powered by Java. USB-650 Red Tide Spectrometer that have a wavelength range of 380 nanometer (nm) to 1000nm and sensitivity of 400 photons/ 400 nm will be used to measure the visible spectra of bioluminescence and whitening product. II. Materials and Methods The different laboratory apparatus that is used in the study where gathered from Pacita Complex National High School Laboratory, San Pedro, Laguna, Philippines and the spectrometer where gathered from the University of the Philippines, Los Banos, Laguna, Philippines while the different chemicals involve in making the luminescent agar were bought from DKL Laboratory, Espanya Street, Manila, Philippines. Varieties of whitening products were obtained randomly from shopping centers from different manufacturing companies and were labelled as brand X, Y, and Z. The whitening product samples were brought on Adamson University Technological and Research Developmental Center (AUTRDC) and undergo Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). The whitening product samples also undergo serial dilution to get the concentration of 0.01%. The visible spectra of whitening product samples with 0.01 v/v concentration was get using USB-650 Red Tide Spectrometer.

6

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

The luminescent bacteria (Aliivibrio fischeri) were isolated from the squid. The squid was bought at the San Pedro Public Market; Laguna, Philippines early in the morning from 5:00 a.m. to 7:00 a.m. to obtain freshly caught samples. The ink of the squid was streaked by multiple times on a Tryptone Yeast Extract Glycerol Salt Water Plate and incubated at 24°C for 10 hours. The Tryptone Yeast Extract Glycerol Salt Water Plate was made of 750ml Artificial Sea Water, 20 g Tryptone Powder, 5g Yeast Extract and 15ml of Glycerol. The formulation of artificial sea water is based from DSM (1993). To prepare a solid medium, agar is added at 18.0 g per dm3, after all the components were dissolved and the pH has been adjusted to 6 using 1 M of hydrochloric acid and 1 M of Sodium Hydroxide Solution, culture media was sterilized on a hot plate at 110°C. A brightly luminous colony from the species was chosen from the plate, fished out using inoculating loop and was inoculated into a freshly prepared artificial sea water , 250 ml in Erlenmeyer flask. 4ml of bioluminescent bacteria and 4 drops of each whitening product sample was placed in different test tube containing 5mL artificial sea water. Every 5 minutes the test tubes were photographed using Canon 700D in manual setting with a 30 seconds exposure. The picture taken was used in the image processing program ImageJ to get the intensity measurement of the bioluminescence intensity. The test tubes also undergo spectrometry. The intensity of the light was measured using an image processing program while the visible spectra was measured using a spectrometer. The image processing program that was used is ImageJ that is powered by Java. USB-650 Red Tide Spectrometer that

7

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

have a wavelength range of 380 nanometer (nm) to 1000nm and sensitivity of 400 photons/ 400 nm was used Figure 1. Flowchart of the Methodology

Preparation of Whitening Products for Spectrometry Preparation of Luminescent Bacteria (Aliivibrio fischeri) Bioluminescence Phenomenon

Preparation of Whitening Product Samples for Rapid Bioluminescence Method

Observation and Interpretation of Results

Research Instrument Table 1. The Amount of Mercury Content of Whitening Product Samples (Brand X, Y, and Z) The Amount of Mercury Content of Whitening Product Samples by ICP OES Brands

Amount (percentage %)

8

9

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Table 1 will show the amount of mercury present in each whitening product samples using the Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). Table 2. Intensity of Light produced by the Bioluminescence in Whitening Product Samples (Brand X, Y, and Z) Intensity of Light produced by the Bioluminescence in Whitening Product Samples Time (Minutes)

X

Y

Z

0 5 10 15 20 25 30 35 40 45 50 Mean

The table shows the intensity of light by Bioluminescent Aliivibrio fischeri in minutes. The means will be used for comparison of intensity of light of bioluminescence on whitening product samples to the amount of mercury content.

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Table 3 . Rate of Decay in Intensity RATE OF DECAY IN INTENSITY X Y Z Table 3 will show the rate of decay or how fast the intensity of light produced by bioluminescent bacteria decrease. This will also show the effect of whitening product samples on the intensity of light by bioluminescence. Risk and Safety The researchers will conduct a study about the bioluminescence method as a screening test of mercury on whitening products with the following risk and safety: 1. Safety measures are needed on the acquisition of whitening products that contain mercury. 2. Use of laboratory gown and hand gloves while handling the bacterial strain. 3. Sterilize equipment and materials before and after using it. 4. Label every materials used in the study.. 5. Autoclave or disinfect all waste materials and disposed it properly. Data Analysis The following statistical treatments were used in the study: 1. The Normality Test was used to determine if the data on the intensity of light of bioluminescence is normally distributed.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

2. The Analysis of Variance (ANOVA) was used to compare the intensity of light of bioluminescence between the whitening product samples.

𝐹=

𝑚𝑒𝑎𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝑓𝑜𝑟 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡𝑠 (𝑀𝑆𝑇) 𝑚𝑒𝑎𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝑓𝑜𝑟 𝑒𝑟𝑟𝑜𝑟𝑠 (𝑀𝑆𝐸)

3. The Pearson Correlation was used to determine if an association exists between the intensity of light produced by the bioluminescent bacteria and the amount of mercury of whitening product sample. The Pearson coefficient was interpreted as follows:

R 0.00

Interpretation/ Conclusion no correlation; no association

±0.01 to ± 0.20

very low correlation; negligible association

± 0.21 to ± 0.40

low correlation; weak association

± 0.41 to ± 0.60

moderate correlation; moderate association

± 0.61 to ± 0.80

high correlation; strong association

± 0.81 to ± 0.99

very high correlation; very strong association

±1.00

perfect correlation; perfect association

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III. Results and Discussion This part of the research discusses the results and findings from the various experiments and tests conducted as described in the methodology. A. Amount of Mercury Present on Each Whitening Product Samples (Brand X, Y, and Z) To get the mercury content of each whitening product samples, the researchers brought the whitening product samples on Adamson University Technological and Research Development Center (AUTRDC) for Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES). (see Appendix B) Table 1. The Amount of Mercury Content of Whitening Product Samples (Brand X, Y, and Z) The Amount of Mercury Content of Whitening Product Samples by ICP OES Brands

Amount (percentage %)

X

0.589

Y

0.229

Z

0.182

Table 1 presents the mercury content of whitening product samples used in the study. As shown, brand X has the highest mercury content at 0.589 %, followed by brand Y at 0.229 % and by brand Z at 0.182%. The results of the study show that brand X has the highest content of mercury. This supports the findings of Food and Drug

12

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Administration (FDA) that cosmetics should have contain mercury at most of 1 part per million (0.0001 %) and if the cosmetics is intended for use only in the area of the eye it contains no more than 65 parts per million (0.0065 %). These whitening products were just some of the harmful whitening products around San Pedro. B. Visible Spectra of Whitening Products Samples in 0.01 v/v Concentration In measuring the visible spectra, the researchers used the method of spectrometry using USB-650 Red Tide Spectrometer by Ocean Optics that can detect a wavelength of 380 nanometer (nm) to 1000nm and sensitivity of 400 photons/count at 400 nm.

Chart 1. Visible Spectra of Brand X in 0.01 v/v Concentration 0.4

Relative Itensity (%)

0.35 0.3 light source

0.25 0.2

Brand X

0.15 0.1 0.05 0 300

400

500

600

700

800

Wavelength ,λ (nm)

The graph shows the visible spectra of brand X in 0.01 v/v concentration. The relative intensity of brand X is relatively high ranging from 0% to 0.37% as compared

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

to the light source ranging from 0% to 0.25% that indicates that the brand X have higher photons than the light source. This shows that the visible spectra of brand X can be seen on the wavelength ranging from 400 nm to 800 nm that have its peak on 550 nm to 650 nm that is color yellow to red.

Chart 2. Visible Spectra of Brand Y in 0.01 v/v concentration 0.45

Relative Intensity (%)

0.4

0.35 0.3

light source

0.25

Brand Y

0.2

0.15 0.1 0.05 0 300

400

500 600 Wavelength λ (nm)

700

800

Chart 2 shows the visible spectra of brand Y in 0.01 v/v concentration. The relative intensity of brand Y is relatively high ranging from 0 to 0.42 as compared to the light source ranging from 0 to 0.25 that indicates that the brand Y have higher photons than the light source and brand X. This shows that the visible spectra of brand Y can be seen on the wavelength ranging from 400 to 800 nm that have its peak on 550 to 650 nanometer (nm) that is color yellow to red.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Chart 3. Visible Spectra of Brand Z in 0.01 v/v Concentration 0.3

Relative Intensity (%)

0.25 light source

0.2

Brand Z

0.15 0.1 0.05 0 300

400

500 600 Wavelength λ (nm)

700

800

Chart 3 shows the visible spectra of brand Z in 0.01 v/v concentration. The relative intensity of brand Z is ranging from 0% to 0.19% as compared to the light source which is relatively high ranging from 0% to 0.25%. The brand Z have lower photons than the light source and brand X and Y. This shows that the visible spectra of brand Z can be seen on the wavelength ranging from 400nm to 800 nm that have its peak on 550 nm to 650 nm like Brand X and Y that is color yellow to red.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

0.45

Chart 4. Visible Spectra of Whitening Product Samples in 0.01 v/v Concentration

0.4

Relative Intnsity (%)

0.35 0.3

light source

0.25

Brand X

0.2 Brand Y

0.15

Brand Z

0.1 0.05 0 300

500 Wavelength λ (nm)

700

Chart 4 shows the visible spectra of Brand X, Y and Z in 0.01 v/v concentrations. The relative intensity of brand X is relatively high ranging from 0% to 0.37% , Brand Y is ranging from 0% to 0.42% compared to the light source ranging from 0% to 0.25%. Brand Z have a relative intensity ranging from 0% to 0.19% which is relatively low compared to light source that indicates that brand Z have lower photons than brand X and Y. If there is a high relative intensity, there is a high photons and if there is a low relative intensity, there is a low photons (Heile, 2013). The brand X and Y have high photons compare to brand Z.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

C. Visible Spectra of Bioluminescence After 50 Minutes Exposure in Whitening Product Samples in 0.01 v/v Concentration

Relative Intensity (%)

Chart 5. Visible Spectra of Bioluminescence after 50 minutes Exposure in Brand X

0.9

bacteria only 0.01 X 0.7

0.5 500

520

540

560

580

600

Wavelength λ (nm)

Chart 5 shows that the bioluminescence with brand X relative intensity decrease by 0.13% to 0.01% (see Appendix C) in respect to the bacteria only that indicates the number of photons also decreases. This means that the Brand X have an effect in the light and metabolism of bioluminescence.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Realtive Intensity (%)

Chart 6. Visible Spectra of Bioluminescence after 50 minutes Exposure in Brand Y

0.9 bacteria only

0.7

0.0 1 Y

0.5 500

520

540

560

580

600

Wavelength λ (nm)

Chart 6 shows that the bioluminescence after 50 minutes exposure to brand Y decreases by 0.22% to 0.1 % (see Appendix C) that also indicate that the number of photons decreases. This stated that the Brand Y of whitening products affects in the light and metabolism of the bioluminescence.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Chart 7. Visible Spectra of Bioluminescence after 50 minutes Exposure in Brand Z

Relative Intensity (%)

0.9

bacteria only

0.7 0.01 Z

0.5 500

520

540

560

580

600

Wavelength λ (nm)

Chart 7shows that the bioluminescence after 50 minutes exposure to brand Z decreases by 0.16 % to 0.1% (see Appendix C) that also indicates that the number of photons decreases. This stated that the Brand Z of whitening products affects in the light and metabolism of the bioluminescence.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Relative Intensity (%)

Chart 8. Visible Spectra of Bioluminescence after 50 minutes Exposure in Whitening Product Samples

0.9 bacteria only 0.01 X 0.7

0.0 1 Y 0.01 Z 0.5 500

520

540

560

580

600

Wavelength λ (nm)

The chart 8 shows the visible spectra of bioluminescence after 50 minutes exposure in whitening product samples. The relative intensity of Brand X, Y, and Z decreases by 0.13% to 0.01%, 0.22% to 0.1 %, and 0.16 % to 0.1% respectively (see Appendix C) that shows that the intensity of light by bioluminescence decreases. The working theory of the bioluminescent bacterium toxicity test system is that the intensity of the light emitted by bacteria faded negatively and correlated to the toxicity of the tested agents. The reduction of light emitted is proportional to the toxic content of the aquatic sample (Alba et.al, 2009).

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

D. Intensity Measurement of Bioluminescence Every 5 Minutes in Whitening Product Samples at 0.01 v/v Concentration Every 5 minutes the test tubes were photographed using Canon 700D in manual setting with a 30 seconds exposure. The photo was used to measure the intensity of the using an image processing program, ImageJ was powered by Java. Table 2. Intensity Measurement of Bioluminescence in Whitening Products Sample every 5 minutes.

Intensity Measurement of Bioluminescence in 50 minutes in Whitening Product Samples Time (min)

X

Y

Z

0

1

1

1

5

0.97323254

0.97402635

0.97426502

10

0.93059927

0.92425544

0.92762392

15

0.89974368

0.89206758

0.87544383

20

0.87745368

0.89435853

0.8937935

25

0.87959508

0.89243986

0.89768499

30

0.8444567

0.85246277

0.85144156

35

0.78751501

0.81248568

0.81585002

40

0.78196684

0.80538373

0.79429058

45

0.76639304

0.76612257

0.77710552

50

0.7402096

0.73694158

0.75665388

Mean

0.861924

0.868231

0.869468

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Table 2 shows the intensity of light of bioluminescence in whitening product samples every 5 minutes. The raw data was normalized so that the data started with a margin of 1 and to avoid the data anomaly (Karwin, 2016) (for the raw data, See Appendix C). The intensity of light produces by the bioluminescence decreases as the time also decreases. The decrease in Aliivibrio fischeri bioluminescence was correlated to the presence of contamination. (Boyton, 2009).

Chart 9. Intensity Measurement of Bioluminescence after 50 Minutes 1

Luminous Intensity (cd)

0.95

X

0.9 Y

0.85

y = -0.0051x + 0.9905 R² = 0.9793

0.8

Z

y = -0.0049x + 0.9912 R² = 0.9708

0.75

Linear (X) Linear (Y) Linear (Z)

0.7 y = -0.0047x + 0.9861 R² = 0.9518

0.65

0.6 0

10

20

30

40

50

60

Time (Minutes)

Chart 9 shows the emitted light intensity is linearly related to time. The y-intercept of the linear fit is 0.9905, 0.9912, and 0.9861 as for brands X, Y, and Z respectively. This is the time at which the luminous lights started to decrease. This shows that the whitening products samples have an effect on the bioluminescent bacteria in terms of the intensity of light.

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Table 3. Rate of Decay in Intensity RATE OF DECAY IN INTENSITY X

-0.005148

Y

-0.004917

Z

-0.004667

Table 3 shows the rate of how fast the intensity of light decreases. Brand X is the fastest while brand Z is the slowest. Luminous bacteria emitting high and steady levels of luminescence under proper conditions are of great interest because of their unique trait of toxicity assessment (Sasaki,et al. ,2009). Since decay of light by the bioluminescent bacteria is affected, this indicates that the Brand X which have more effect on the metabolism and light produced by the bioluminescent bacteria have the highest contaminants than the brand Y and Z..

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E. Difference in the Intensity of Light of Bioluminescence between the Whitening Product Samples (Brand X, Y, and Z) Table 4. Comparison of Intensity of Light of Bioluminescence in Whitening Product Samples Whitening Product Samples Brand X

N

Mean

11

26.57

Brand Y

11

30.32

Brand Z

11

30.61

F Statistics

p-value

7.232*

0.02732

*Highly Significant at 0 .01

Following the result of the normality test (see appendix D), Table 4 presents the comparison of intensity of light of bioluminescence in whitening product samples. The analysis of variance (ANOVA) revealed that the difference between the intensity of light of bioluminescence in whitening product samples is highly significant (p<0.01). The computed value for F is 7.232 which exceeds Fo.o1 = 5.3905 thereby the null hypothesis is rejected.

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Table 5. Post Hoc Analysis ( Tukey HSD Test) of Intensity of Light of Bioluminescence in Whitening Product Samples

Q Statistics

p – value

Brand Y

4.4750

0.00964

Brand Z

4.8219

0.00517

Brand Z

0.3470

0.89999

Whitening Product Samples Brand X

Brand Y

*Highly Significant at 0.01

Based on the result of the ANOVA, a post analysis was conducted and the summary is shown on Table 5. Between brand Y and brand Z, the Q statistics is not significant (p>0.01); between brand X and brand Z, the Q statistics is highly significant (p<0.01); between brand X and brand Y, the Q statistics is highly significant (p<0.01). It indicates that the mean difference between brand X and brand Y and between brand X and brand Z is significant while the mean difference between brand Y and brand Z is not significant. Therefore, brand X (highest mercury content) has a significantly lower intensity of light of bioluminescence compared to brand Y and brand Z (lowest mercury contents).

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

F. Association of Intensity of Light produced by the Bioluminescence in Mercury Content of Whitening Product Samples (Brand X, Y, and Z) Table 7. Correlation between Intensity of Light of Bioluminescence and Mercury Content in Whitening Product Samples

Variables

Pearson Coefficient

p-value

Amount of Mercury on Whitening Products and Intensity of Light produced by Bioluminescence

-0.9991*

0.02847

* Highly Significant at 0.05

Table 7 shows that the correlation between the amount of mercury content in each brand and intensity of light produced by bioluminescence has very high correlation; very strong association with R is -0.9991 as stipulated in Pearson R correlation interpretation table and highly significant at p < 0.05. Pearson coefficient is negative that indicate that the two variables have a negative correlation. The variables show an inverse proportion, as the amount of mercury increases, the intensity of light decreases.

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

IV. Conclusion and Recommendation This shows the conclusion and findings gathered of this study.

Conclusion 1. Brand X have the highest mercury content with 0.589 % while Brand Z have the lowest mercury content with 0.182% based on the result of ICP OES. But still, they both surpass the FDA regulated amount of mercury on whitening products. 2. Brand Y has the highest related intensity ranging from 0% to 0.42% . Brand Z have a relative intensity ranging from 0% to 0.19% which is relatively low compared to light source. If there is a high relative intensity, there is a high photons and if there is a low relative intensity, there is a low photons. 3. The relative intensity of Brand X, Y, and Z decreases by 0.13% to 0.01%, 0.22% to 0.1 %, and 0.16 % to 0.1% respectively (see Appendix C) that shows that the intensity of light by bioluminescence decreases. 4. Brand X is the fastest while brand Z is the slowest. Since decay of light by the bioluminescent bacteria is affected, this indicates that the Brand X which have more effect on the metabolism and light produced by the bioluminescent bacteria have the highest contaminants than the brand Y and Z.

5. The analysis of variance (ANOVA) revealed that the difference in intensity of light of bioluminescence is highly significant (p<0.01) Intensity of light of bioluminescence differs significantly across whitening products with different mercury content. 6. The correlation between the amount of mercury content in each brand and intensity of light produced by bioluminescence has very high correlation; very

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BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

strong association with R is -0.9991 as stipulated in Pearson R correlation interpretation table and highly significant (p < 0.05). As the mercury content of whitening product increases the luminous light and the rate of decay of the bioluminescence decreases and vice versa. Recommendations Based on the results and findings, the following recommendations are offered: 1. Have the further study on the luminescent bacteria, which is the Aliivibrio fischeri, bioluminescence method and the mercury. 2. The danger of mercury is of great concern in the public. The ability to detect hazardous chemical such as those tested in this experiment is crucial to the public health. X-ray Fluorescent (XRF) can be used as a screening in quantitative testing in detecting mercury. 3. Use DNA Barcode or Meta barcode for the verification of the Aliivibrio fischeri.

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Bibliography Formoso, C. (2017). Poisoned lake, quaysides threaten residents in 2 Puerto Princesa villages. Philippine News Agency. Retrieved June 7, 2017, from http://www.pna.gov.ph/articles/994121 Simoune, A. (2016). Mercury Poisoning Linked to Skin Products. Retrieved August 25, 2017, from https://www.fda.gov/ForConsumers/ConsumerUpdates/ucm294849.htm U.S. Food and Drug Administration. (n.d.). CFR - Code of Federal Regulations Title 21. Retrieved August 25, 2017, from https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=700.1 3 Quinto, E. (2013) Simple water toxicity test using Photobacterium leiognathi Retrieved August 25, 2017, from http://web.a.ebscohost.com/ehost/detail/detail?sid=6e0c495685104974920181828c 569ac9%40sessionmgr4005&vid=0&hid=4209&bdata=JnNpdGU9ZWhvc3QtbGl2 ZQ%3d%3d World Health Organization. (n.d.). International Programme on Chemical Safety, Mercury. Retrieved September 2, 2017, from http://www.who.int/ipcs/assessment/public_health/mercury/en/ CrawLaw. (n.d.). Philippine Environmental Laws – Chan Robles Virtual Law Library. Retrieved September 2, 2017, from http://www.chanrobles.com/presidentialdecreeno856.htm#.WaoyrYVOLIU

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Case J., Haddock S., and Moline M. (2010) Bioluminescence in the Sea. Retrieved September 2, 2017, from https://www.researchgate.net/publication/49667852 Suvarapu L. and Sungok B. (2015).Recent Developments in the Speciation and Determination of Mercury (Hg) Using Various Analytical Techniques. Retrieved September 4, 2017, from https://www.hindawi.com/journals/jamc/372459/ Lu J. (2014). Cyanide and Mercury Level Determination in Small Scale Mining Areas in the Philippines. Retrieved September 2, 2017, from www.tsijournals.com

Naguit M., Abisado R., Plata K., et.al (2014).Evidence of Bacterial Bioluminescence in a Philippine Squid and Octopus Hosts. Retrieved September 2, 2017, from http://www.bioflux.com.ro/aacl

Sasaki, S., Okamoto, T. and Fujii, T. (2009) Bioluminescence intensity difference observed in luminous bacteria groups with different motility. Lett Appl Microbiol 48, 313–317. Retrieved: September 10, 2017 From: http:// onlinelibrary.wiley.com/doi/10.1111/j.1472-765X.2008.02525.x/full

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ACKNOWLEDGEMENT This research study was a milestone to their journey, and is more astonishing because of those souls who served as their sources of inspiration that lead the researchers made it successfully. In this manner, the researchers would like to express their gratitude to the following individuals: The Almighty God, who guide the researchers in every steps of their life and the ultimate source of all his wisdom, forgiveness, and blessings, Mrs. Sonia J. Alvero for her unquestionable hope and support to the researchers that led to the success of this research work, Their research mentor and research adviser Ms. Clarence V. Bellen, for her incomparable assistance that guided the researchers in finishing the study, Their ever-supportive adviser, Ms. Irma Estela Marie L. Esteban and the rest of their subject teachers, for their kind consideration, Their classmates, King Ace M. Anievas, Mary Antonette O. Irarum, and Mary Lorraine O. Irarum of 10-Einstein also Ma. Elijah Callanta, Cale Herrera, Jewel Olila and Siobe Veloso of 10 - Newton for their undeniable helpfulness in whatever the researcher asked them to do, The researchers’ Alma Mater, Pacita Complex National High School, specifically the Science department, headed by Mrs. Maria Belynda L. Lallabban, for the encouragement and reassurance to finish this study,

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Mr. Emmanuel Joshua G. Mohammetano, for advising the researchers on their research procedure and actual experimentations, Mr. Emmanuel Louie Aribon, for accompanying the researchers for their travel on finding chemicals and bacterial strain on Manila. Dr. Darwin B. Putungan, head of Physics Division of University of the Philippines Los Baños for allowing the researchers to use their laboratory. Prof. Lou Serafin Lozada, professor in University of the Philippines Los Baños for being generous in his knowledge, for helping the researchers to conduct their experimentation and for teaching the researchers about the Science of Spectrometry and allotting his time to help them do their research. The researchers’ friends for their moral support as they conduct their study, The researchers’ beloved family who inspired and encouraged them, for their support physically, emotionally, morally, spiritually, and most of all, financially in accomplishing this study.

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

DEDICATION The researchers’ dedicate this research work to their ever supportive and beloved parents, friends, teachers, and classmates, who gave their all-out support, encouragement and inspiration, and most of all, to the one responsible for all the great things they have accomplished, the Almighty God.

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Appendix A: Photo Documentation Preparation of Whitening Products for Spectrometry

Preparation of Luminescent Bacteria (Aliivibrio fischeri)

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Bioluminescence Phenomenon

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Preparation of Whitening Product Samples for Rapid Bioluminescence Method

Observation and Interpretation of Results

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Appendix B: Results and Certification

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Appendix C: Tables and Charts Table 1.Visible Spectra of Whitening Products Samples in 0.01 v/v Concentration Wavelength (λ) 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438

light source 0.03229689 0.03179745 0.03594537 0.03344815 0.03459941 0.03594961 0.04061814 0.03684268 0.03685115 0.03803627 0.03887432 0.04118107 0.04050809 0.04049963 0.04079591 0.04265401 0.04357248 0.04486765 0.04295876 0.04615436 0.04486765 0.04561259 0.04769501 0.05660036 0.04844841 0.0488759 0.05076787 0.05059856 0.05220271 0.0519022 0.05309579 0.0539423 0.05500891 0.05618557 0.05621096 0.05658343 0.05808176 0.05812409 0.06049433

Brand X 0.0122 0.011993 0.015779 0.013266 0.013461 0.014328 0.01836 0.014197 0.014125 0.015026 0.014908 0.016866 0.016287 0.015864 0.016253 0.017882 0.019341 0.020712 0.018631 0.022058 0.021398 0.022908 0.024029 0.034039 0.026635 0.026208 0.028446 0.028209 0.029597 0.028776 0.029123 0.029605 0.030282 0.031424 0.03057 0.030853 0.032135 0.032465 0.034614

Brand Y 0.007989 0.007761 0.011119 0.008505 0.008657 0.009613 0.013436 0.009224 0.009435 0.00985 0.009638 0.011618 0.010746 0.01018 0.010408 0.011719 0.01347 0.014519 0.012075 0.015094 0.014122 0.014676 0.016422 0.025355 0.01742 0.016947 0.01907 0.018309 0.01937 0.01885 0.019315 0.019933 0.020182 0.021261 0.020085 0.020664 0.022014 0.02154 0.023595

Brand Z 0.003972 0.003596 0.006772 0.004217 0.004234 0.004877 0.008734 0.004412 0.004395 0.004784 0.004835 0.006463 0.005292 0.004623 0.004522 0.005829 0.006991 0.007406 0.004979 0.007727 0.005926 0.006171 0.007676 0.015863 0.007566 0.00692 0.008598 0.007854 0.008746 0.007321 0.007972 0.00859 0.008632 0.009689 0.007854 0.007896 0.00963 0.008564 0.01056

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

0.059542 0.06157787 0.06182336 0.06258946 0.06503166 0.06341481 0.06498087 0.0651544 0.06710562 0.06928117 0.06964517 0.06935736 0.07089801 0.07329365 0.07439836 0.07491897 0.07629032 0.07822884 0.07896954 0.07754316 0.07926159 0.08073453 0.08039169 0.08302012 0.08382431 0.08291854 0.08451846 0.08605065 0.08731196 0.08910657 0.08825159 0.09113821 0.09362697 0.09297515 0.09367352 0.09445655 0.09840978 0.10447084 0.104492 0.10218524 0.1028032 0.10579987 0.10499991 0.1078315 0.10978696

0.033265 0.03601 0.037085 0.038557 0.041019 0.04041 0.042255 0.04352 0.045698 0.04822 0.049252 0.04899 0.051604 0.052899 0.053534 0.053754 0.054143 0.056453 0.057125 0.054701 0.05586 0.056174 0.056262 0.05707 0.057883 0.057197 0.059207 0.060557 0.061775 0.064279 0.063928 0.067402 0.071167 0.072157 0.074361 0.077327 0.083512 0.092862 0.094732 0.09403 0.098446 0.105452 0.10684 0.112509 0.116469

0.022695 0.024441 0.025109 0.026488 0.028984 0.027148 0.028984 0.029648 0.031352 0.034127 0.03438 0.034393 0.036258 0.037679 0.038212 0.037865 0.03883 0.04108 0.041638 0.039016 0.040496 0.040741 0.040394 0.042306 0.042898 0.041832 0.043833 0.045165 0.046316 0.047948 0.047424 0.050765 0.054191 0.054318 0.055506 0.05853 0.064891 0.073485 0.074792 0.073443 0.077055 0.082934 0.08564 0.089506 0.093143

0.008784 0.010129 0.010374 0.011055 0.013013 0.010628 0.011051 0.011182 0.012734 0.013935 0.013351 0.012971 0.014138 0.014569 0.014861 0.014298 0.015169 0.016421 0.017132 0.014797 0.01544 0.015871 0.015102 0.016802 0.017233 0.015863 0.016865 0.017444 0.017901 0.019381 0.017779 0.020548 0.021639 0.020726 0.021229 0.021935 0.026088 0.032541 0.032951 0.029022 0.029767 0.032989 0.033268 0.035247 0.036109

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528

0.11726169 0.11379944 0.11569564 0.11707546 0.12144348 0.12634481 0.12259474 0.12519354 0.12658183 0.12685272 0.12896901 0.13274447 0.13132232 0.13316349 0.13436978 0.13497927 0.13490308 0.1364903 0.13767965 0.13989753 0.15091916 0.14248786 0.14259791 0.14495969 0.14691514 0.14854892 0.14880711 0.15045358 0.1525868 0.15565119 0.15534644 0.15712412 0.16337988 0.1591219 0.16337141 0.16470467 0.16564854 0.17098159 0.17466393 0.173919 0.18159267 0.18241802 0.17641622 0.1819609 0.19402375

0.125717 0.123987 0.127325 0.131022 0.137072 0.142365 0.139831 0.142868 0.144264 0.145944 0.1483 0.15281 0.152345 0.153699 0.156 0.158124 0.159503 0.162685 0.16414 0.166907 0.17838 0.172026 0.172813 0.176883 0.180783 0.184219 0.186393 0.190912 0.195049 0.199923 0.202334 0.207843 0.216609 0.213393 0.222565 0.227084 0.229766 0.237508 0.242094 0.246756 0.261022 0.263772 0.260261 0.267377 0.289334

0.102905 0.101285 0.104402 0.108682 0.114595 0.119975 0.117809 0.121269 0.123071 0.125164 0.127478 0.131969 0.132231 0.134718 0.136723 0.139768 0.141079 0.143892 0.146603 0.149479 0.161617 0.154283 0.156605 0.159866 0.164603 0.167488 0.170473 0.174288 0.177858 0.183289 0.184693 0.189544 0.199521 0.196696 0.2043 0.209553 0.211744 0.220008 0.223662 0.228721 0.242783 0.245427 0.242365 0.249724 0.272935

0.042748 0.039535 0.039924 0.042376 0.04652 0.049899 0.046529 0.048212 0.048449 0.049709 0.050461 0.054318 0.052939 0.054047 0.055764 0.056931 0.056745 0.058716 0.05979 0.061058 0.067274 0.063883 0.064094 0.066259 0.068898 0.070674 0.071021 0.07349 0.075571 0.078488 0.078319 0.081157 0.087918 0.083943 0.088493 0.090798 0.090811 0.095978 0.096105 0.098938 0.109941 0.11022 0.104646 0.110651 0.130669

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573

0.18273546 0.1862612 0.18852986 0.18854679 0.18722623 0.19030331 0.19250002 0.19258467 0.19332961 0.19536125 0.19648711 0.19794312 0.19862033 0.19977159 0.19741828 0.19870922 0.19881503 0.19985625 0.20316612 0.2059173 0.20749605 0.21528823 0.20775424 0.20436818 0.20805899 0.20978165 0.21361213 0.21227463 0.21370525 0.21517818 0.21848806 0.22076519 0.21992714 0.22078212 0.21946155 0.23804258 0.22851081 0.22312697 0.22450679 0.23009379 0.22965361 0.22929807 0.23087259 0.22879016 0.23393698

0.272331 0.276016 0.276498 0.276346 0.275897 0.277814 0.278715 0.279265 0.279087 0.278926 0.279844 0.279992 0.279079 0.279595 0.278943 0.279806 0.27984 0.280805 0.281279 0.279823 0.280669 0.289918 0.284257 0.284316 0.286905 0.287942 0.289613 0.290104 0.291703 0.295984 0.299022 0.301471 0.302271 0.307619 0.309751 0.330016 0.32301 0.322113 0.324321 0.329423 0.334094 0.338875 0.342606 0.344341 0.348588

0.254025 0.258216 0.258927 0.259849 0.259443 0.260991 0.264171 0.263309 0.264366 0.264349 0.266942 0.265922 0.266388 0.268798 0.267352 0.269644 0.26967 0.270524 0.27071 0.270279 0.272258 0.280108 0.275633 0.275379 0.278805 0.278805 0.28152 0.282104 0.284921 0.288871 0.292838 0.296175 0.296831 0.302574 0.303877 0.32636 0.318811 0.31873 0.323823 0.327731 0.332036 0.339048 0.34255 0.344496 0.349563

0.112288 0.114584 0.114296 0.114322 0.113425 0.114305 0.115641 0.116436 0.116419 0.116038 0.117933 0.117112 0.118381 0.118685 0.118119 0.119468 0.118905 0.120512 0.120385 0.119633 0.121079 0.129299 0.123574 0.121984 0.123303 0.124174 0.125443 0.12617 0.126847 0.129029 0.129993 0.132014 0.131557 0.135422 0.135253 0.153681 0.143266 0.142585 0.143093 0.145495 0.147922 0.151212 0.153833 0.152929 0.155576

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618

0.23816956 0.23554536 0.23770397 0.23677704 0.23691671 0.24087841 0.24147097 0.24703258 0.24484857 0.24191116 0.24009115 0.24395126 0.24304549 0.24031124 0.24086994 0.24219897 0.24156408 0.24166567 0.24459461 0.24594057 0.24470466 0.24564429 0.24315553 0.24378619 0.2431386 0.24580513 0.24135246 0.23866053 0.23744155 0.24795528 0.24371 0.24026045 0.23944779 0.24120008 0.2366035 0.23747541 0.24002343 0.24142018 0.2414498 0.24209739 0.23958324 0.23695904 0.23411474 0.23343753 0.23478349

0.351093 0.356229 0.358996 0.361048 0.362008 0.363946 0.365435 0.369352 0.363895 0.364682 0.362025 0.365985 0.360104 0.359706 0.356728 0.356534 0.356669 0.354807 0.35298 0.351973 0.351271 0.349671 0.347277 0.347556 0.344637 0.348428 0.342953 0.343732 0.342598 0.348309 0.343812 0.344104 0.344087 0.343816 0.343461 0.345635 0.34363 0.349925 0.352988 0.351211 0.350949 0.349595 0.352269 0.353944 0.355383

0.353894 0.358588 0.363063 0.364881 0.366176 0.370194 0.372283 0.378373 0.371361 0.373535 0.371606 0.374897 0.369212 0.368789 0.366074 0.368806 0.368155 0.36703 0.365617 0.36599 0.366379 0.363452 0.361676 0.360694 0.360864 0.363841 0.360204 0.360136 0.36039 0.366751 0.361506 0.36187 0.363004 0.3639 0.363393 0.36637 0.366032 0.374423 0.376123 0.374778 0.374931 0.374516 0.377722 0.380776 0.381664

0.157479 0.160007 0.161259 0.162853 0.163652 0.165428 0.166485 0.170765 0.165225 0.166198 0.165517 0.167568 0.164151 0.165555 0.162933 0.164236 0.164287 0.163466 0.163094 0.163754 0.163576 0.162984 0.162122 0.161128 0.161513 0.164692 0.161149 0.161293 0.160642 0.167111 0.162028 0.163145 0.163297 0.164151 0.163238 0.165259 0.163356 0.17112 0.172672 0.16997 0.169386 0.168075 0.169944 0.171348 0.172921

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663

0.24085301 0.23230743 0.23072022 0.22972133 0.22840076 0.23139743 0.2281722 0.22811295 0.22711406 0.23393274 0.23246404 0.2246507 0.22517554 0.22496391 0.22487926 0.22096835 0.21956314 0.2173114 0.22220427 0.21927955 0.21904676 0.21636331 0.21362059 0.2145179 0.21186831 0.20971816 0.21119109 0.20661991 0.20437664 0.20581995 0.19954304 0.19832405 0.19631781 0.19591995 0.19371054 0.19125564 0.18722623 0.18568557 0.18395868 0.18097894 0.1804795 0.17877377 0.17876954 0.17656859 0.17623845

0.363108 0.357849 0.360096 0.360908 0.362423 0.369158 0.366983 0.369149 0.369403 0.37888 0.377543 0.370715 0.372974 0.373617 0.374717 0.370723 0.371629 0.369022 0.371028 0.367584 0.365613 0.362321 0.361466 0.360595 0.356424 0.352743 0.350281 0.345678 0.342648 0.343469 0.337233 0.334407 0.332495 0.33153 0.328594 0.326479 0.319794 0.317967 0.315835 0.312653 0.311333 0.309962 0.308092 0.306222 0.305131

0.39074 0.385792 0.389446 0.391264 0.392263 0.400366 0.399258 0.402726 0.403673 0.413947 0.412606 0.406067 0.409493 0.411752 0.412327 0.409552 0.40984 0.408969 0.411109 0.408199 0.404333 0.404367 0.402362 0.40177 0.397921 0.394191 0.393125 0.388245 0.384557 0.386426 0.380141 0.377519 0.37515 0.374135 0.371014 0.369069 0.362843 0.362132 0.358597 0.356643 0.35638 0.353373 0.350442 0.349664 0.349241

0.179078 0.173522 0.175349 0.175289 0.176761 0.181252 0.180245 0.181827 0.181734 0.191092 0.188203 0.183763 0.185125 0.18581 0.187552 0.184398 0.18488 0.184964 0.187958 0.18466 0.183645 0.182478 0.182241 0.184432 0.179839 0.17775 0.178029 0.175154 0.175002 0.176681 0.172659 0.171331 0.16909 0.17057 0.168735 0.168515 0.165048 0.163289 0.162688 0.161335 0.16054 0.160493 0.159686 0.158637 0.158637

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708

0.17691567 0.17326718 0.17236988 0.17120168 0.16994884 0.17053294 0.1677225 0.16929702 0.16676594 0.16537765 0.16432797 0.16431104 0.16521682 0.16464118 0.165293 0.16519142 0.17012661 0.16667282 0.16144982 0.16617338 0.16096731 0.1619408 0.16150908 0.16315978 0.16208471 0.16014619 0.15971446 0.16053558 0.1598753 0.16005307 0.15966367 0.15889334 0.15810185 0.15922348 0.15958749 0.15755585 0.16623263 0.15736961 0.15775901 0.15601519 0.15943511 0.15597286 0.15422904 0.16056098 0.15228205

0.306248 0.302178 0.299834 0.29826 0.29925 0.29892 0.296365 0.297177 0.295891 0.295849 0.294901 0.293932 0.29595 0.294047 0.29634 0.297008 0.302483 0.297863 0.294334 0.299217 0.295392 0.295214 0.29425 0.296196 0.29656 0.294867 0.294114 0.294935 0.293624 0.293065 0.293184 0.291305 0.289985 0.290704 0.289562 0.286305 0.29463 0.284925 0.281143 0.278326 0.281439 0.274933 0.271523 0.276439 0.266277

0.350578 0.347516 0.34475 0.343574 0.343777 0.343963 0.34288 0.343625 0.343312 0.342364 0.341654 0.342284 0.343625 0.343168 0.346018 0.346704 0.353014 0.348641 0.344648 0.349876 0.346441 0.348784 0.347236 0.350696 0.350552 0.350595 0.3487 0.350908 0.350358 0.349893 0.350595 0.34848 0.348894 0.347439 0.348353 0.344284 0.35331 0.344157 0.340571 0.337281 0.339607 0.334261 0.330201 0.335775 0.324305

0.160768 0.158079 0.157369 0.156337 0.156709 0.158443 0.15599 0.158341 0.156108 0.156151 0.15665 0.156142 0.157335 0.157648 0.159331 0.160134 0.165175 0.162587 0.157893 0.163365 0.159821 0.160836 0.15961 0.161724 0.162105 0.161217 0.160523 0.161978 0.160252 0.160709 0.161656 0.15977 0.158857 0.159838 0.159914 0.158519 0.166807 0.157775 0.157132 0.155407 0.158104 0.154155 0.151339 0.157825 0.149055

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753

0.15460997 0.15066521 0.15065674 0.14966632 0.1490399 0.14773626 0.1468051 0.14639877 0.14646649 0.1443248 0.14357987 0.14217465 0.14026999 0.14018534 0.1405832 0.13951659 0.14115883 0.13644374 0.14419783 0.13375182 0.1332947 0.13119534 0.13074669 0.12947691 0.12721672 0.12664955 0.12664109 0.12419466 0.12436396 0.12308572 0.12198525 0.12436396 0.12019064 0.12602313 0.11782886 0.11690615 0.11655908 0.11918328 0.11582261 0.11710932 0.11355183 0.11369786 0.11125143 0.11300372 0.11237729

0.266818 0.259618 0.258061 0.255446 0.252121 0.247755 0.245606 0.24107 0.240715 0.2348 0.232076 0.229334 0.225806 0.222075 0.221271 0.21924 0.219553 0.212573 0.21957 0.207234 0.204424 0.201124 0.200312 0.197715 0.193882 0.192714 0.190548 0.187366 0.186639 0.184379 0.182188 0.184159 0.178177 0.184083 0.175377 0.175047 0.172356 0.175275 0.171493 0.172331 0.16832 0.167381 0.164969 0.166137 0.164969

0.324119 0.316971 0.315567 0.311972 0.308039 0.304266 0.301068 0.29678 0.296763 0.289378 0.28745 0.283314 0.278813 0.275828 0.274212 0.271454 0.271454 0.264603 0.270507 0.258132 0.253936 0.251534 0.249893 0.247338 0.242094 0.24085 0.238457 0.234456 0.234041 0.231698 0.229262 0.231749 0.226107 0.231453 0.223062 0.221531 0.219357 0.222267 0.217208 0.217927 0.214387 0.214544 0.210602 0.211532 0.21149

0.150239 0.145731 0.146239 0.143287 0.142467 0.139211 0.139042 0.136217 0.137071 0.133012 0.131955 0.130805 0.128166 0.126711 0.126559 0.125806 0.126914 0.122009 0.129274 0.119261 0.117755 0.116224 0.115768 0.113932 0.111979 0.111082 0.110575 0.10885 0.108697 0.107108 0.10545 0.108816 0.103657 0.110211 0.102558 0.102498 0.101061 0.105002 0.101018 0.102524 0.099025 0.099471 0.097077 0.099471 0.097999

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798

0.11486605 0.11059115 0.10910128 0.10874574 0.10966844 0.13789128 0.10784843 0.11194557 0.10676489 0.10751829 0.10607922 0.10588452 0.10606229 0.10614694 0.10470786 0.1052073 0.10491737 0.10337036 0.10391213 0.10356506 0.10276934 0.10282013 0.10217678 0.1018805 0.10180431 0.10124561 0.10234608 0.10323492 0.10696806 0.10222545 0.10052607 0.10318413 0.1001536 0.10614694 0.09967109 0.0990362 0.1013218 0.09919704 0.10066151 0.0981135 0.09783203 0.09866374 0.09780876 0.09908699 0.09836746

0.166755 0.163404 0.1604 0.160654 0.161077 0.180047 0.158623 0.162346 0.15644 0.157566 0.154934 0.154968 0.156017 0.154029 0.15314 0.152996 0.152817 0.150551 0.151 0.150289 0.14891 0.148461 0.147776 0.146566 0.145703 0.144383 0.143807 0.142919 0.145728 0.14253 0.142141 0.145745 0.142843 0.14957 0.14363 0.143621 0.150077 0.143537 0.147006 0.144374 0.144072 0.144958 0.144011 0.145618 0.143791

0.21374 0.20957 0.206694 0.205907 0.207455 0.226834 0.205188 0.207802 0.203505 0.203919 0.201864 0.201044 0.202414 0.201179 0.20117 0.200291 0.199637 0.197922 0.198007 0.197119 0.196738 0.19552 0.194801 0.193507 0.193507 0.190775 0.190292 0.188914 0.192948 0.188415 0.189641 0.194006 0.191003 0.197745 0.194243 0.19453 0.198633 0.195325 0.199386 0.196137 0.196998 0.197584 0.198083 0.197694 0.19673

0.100714 0.097289 0.0958 0.095479 0.096722 0.127642 0.094853 0.099767 0.094769 0.094718 0.093542 0.093491 0.095149 0.094244 0.093195 0.093441 0.093012 0.091648 0.092155 0.091851 0.091885 0.091436 0.090667 0.089999 0.090464 0.08862 0.088637 0.088586 0.092011 0.088635 0.088426 0.09136 0.08972 0.096079 0.090388 0.090616 0.094244 0.091014 0.093804 0.091639 0.091324 0.092214 0.092214 0.093711 0.091699

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

799 0.09695377 0.141557 0.195292 0.090971 800 0.10335343 0.149096 0.201822 0.097238 Table 2. Visible Spectra of Bioluminescence After 50 Minutes Exposure in Whitening Product Samples in 0.01 % v/v Concentration Wavelength (λ) 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

Bacteria Only 0.599414352 0.602785234 0.605669211 0.608241069 0.624783361 0.614995318 0.61641858 0.624209063 0.631737366 0.635208126 0.641793831 0.652474534 0.66211276 0.671651108 0.677081974 0.689791448 0.710815765 0.712201572 0.732539228 0.742770479 0.751828164 0.770605227 0.782428284 0.794126493 0.814457907 0.821967483 0.821205913 0.833902903 0.866113554 0.842042959 0.847473825 0.848547513 0.848859632 0.844652271

Brand X 0.470867 0.477498 0.47582 0.479442 0.53382 0.486366 0.484848 0.496911 0.506178 0.514753 0.520878 0.53153 0.540317 0.555443 0.551608 0.566175 0.606146 0.58628 0.606679 0.61275 0.619141 0.64689 0.65115 0.657701 0.713491 0.716393 0.680496 0.710695 0.777162 0.720601 0.729016 0.725288 0.7214 0.71879

Brand Y 0.382193 0.393437 0.397711 0.39587 0.52001 0.404812 0.398368 0.412176 0.428351 0.433217 0.434072 0.437819 0.447682 0.484898 0.454126 0.471156 0.559724 0.487134 0.515538 0.519089 0.506728 0.5638 0.541839 0.547231 0.668807 0.664664 0.568666 0.623109 0.771709 0.61969 0.641651 0.621794 0.612194 0.589838

Brand Z 0.434417 0.446511 0.447442 0.449651 0.565245 0.454186 0.447732 0.463955 0.484073 0.490469 0.494481 0.497912 0.509425 0.538963 0.516053 0.531288 0.604668 0.54594 0.574199 0.581177 0.574897 0.618041 0.602574 0.615831 0.723343 0.712935 0.634206 0.690723 0.824109 0.683571 0.694677 0.688165 0.677117 0.655487

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578

0.846219107 0.846899526 0.845338933 0.843129132 0.836911727 0.83962716 0.835850524 0.831206197 0.832354794 0.828434583 0.829121244 0.828222342 0.826736657 0.827498227 0.823465653 0.827978889 0.838185172 0.828472037 0.828909003 0.835613313 0.834346111 0.839820674 0.840769515 0.842667196 0.850869676 0.858884885 0.861163352 0.864877564 0.878073944 0.881070283 0.911695372 0.907269529 0.911245921 0.919510825 0.929286383 0.93637772 0.948825015 0.955697869 0.960847828 0.969300003 0.975042987 0.983532616 0.988002156 0.9927963 0.995605368

0.723716 0.725447 0.726939 0.724915 0.723051 0.726992 0.726726 0.727524 0.727844 0.724862 0.732025 0.729016 0.734288 0.735087 0.733916 0.736019 0.780411 0.749787 0.743449 0.752024 0.747923 0.761557 0.761877 0.76225 0.776683 0.782275 0.789865 0.789998 0.814817 0.813965 0.914146 0.861366 0.851246 0.859928 0.863656 0.876438 0.896144 0.902588 0.905944 0.915797 0.925969 0.935556 0.946474 0.948045 0.952546

0.598386 0.620479 0.614035 0.612194 0.604567 0.613378 0.615219 0.619558 0.61443 0.604961 0.623438 0.61443 0.615482 0.628764 0.613378 0.627186 0.715951 0.655196 0.640599 0.644413 0.646122 0.659273 0.655722 0.659404 0.670713 0.665059 0.681431 0.683338 0.703195 0.697146 0.914391 0.776311 0.738701 0.737912 0.746854 0.765396 0.780388 0.79722 0.781045 0.793012 0.799456 0.820891 0.824836 0.830162 0.83299

0.675547 0.678745 0.675722 0.674442 0.669442 0.673628 0.674675 0.674907 0.669791 0.663162 0.674326 0.670837 0.673745 0.681885 0.674442 0.680257 0.76515 0.710725 0.6927 0.701306 0.709213 0.713284 0.707934 0.710376 0.723866 0.720959 0.737123 0.739798 0.760498 0.752358 0.950053 0.830273 0.795734 0.791664 0.807596 0.815039 0.83411 0.84353 0.832831 0.845274 0.848414 0.872254 0.880394 0.874929 0.879464

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600

0.996404392 0.995205856 1 0.991435462 0.990049655 0.980398944 0.981285362 0.972371251 0.973881906 0.967152626 0.961909032 0.958731663 0.954068609 0.947913628 0.942595125 0.938437704 0.933356411 0.927338762 0.923580853 0.918012655 0.917775445 0.90758789

0.957872 0.964316 0.988123 0.950096 0.95899 0.946687 0.964902 0.942213 0.952599 0.941468 0.939284 0.935902 0.929165 0.926768 0.923147 0.923839 0.920004 0.914838 0.907621 0.907222 0.918034 0.900352

0.851926 0.851269 0.911366 0.829702 0.847981 0.83036 0.866918 0.829702 0.848507 0.820628 0.827466 0.818919 0.821943 0.836146 0.828518 0.839039 0.829965 0.82431 0.816157 0.810765 0.850085 0.815763

0.897605 0.894117 0.944703 0.880627 0.89307 0.87301 0.913305 0.876673 0.891209 0.86923 0.875278 0.860101 0.85388 0.871324 0.863416 0.865742 0.858532 0.861323 0.848065 0.843763 0.880278 0.843763

Table 3. Raw Data (Not normalized) of Intensity Measurement of Bioluminescence in 50 minutes in Whitening Product Samples

Time (mins) 0 5 10 15 20 25 30 35 40 45 50

X 30.821 29.996 28.682 27.731 27.044 27.11 26.027 24.272 24.101 23.621 22.814 30.821

Y 34.92 34.013 32.275 31.151 31.231 31.164 29.768 28.372 28.124 26.753 25.734 34.92

Z 35.205 34.299 32.657 30.82 31.466 31.603 29.975 28.722 27.963 27.358 26.638 35.205

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Appendix D: Statistics Table 4. Results of the Normality Test (Shapiro – Wilk Test) Shapiro – Wilk Test

Whitening Intensity of Light of Biolumine scence

Products

W Statistic

df

p-value

X

0.953

3

0.685

Y

0.969

3

0.875

Z

0.965

3

0.833

*Highly Significant at 0.05

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

Data Logbook

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT

BIOLUMINESCENCE METHOD AS A SCREENING TEST OF Hg CONTENT