Cytytoxicity Bench.docx

WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 1 of 10 Page No.

I. RESEARCH TITLE: BRINE SHRIMP LETHALITY ASSAY BENCH II. RESEARCH TYPE: PROJECT III. PROPONENT Mr. Prency D. Yerro Biology Education Email address: [email protected]; cell phone no.: 09772094711 Mr. Christopher Emboltorio Laboratory Technician WVSU - Central Science Laboratory Email address: IV. DURATION OF STUDY Expected Start Date: January, 2019 Expected Completion Date: June, 2021 Total No. of Years/Months: 2 year V. KEYWORDS Cytotoxicity, Brine Shrimp Lethality Assay, Environmental Factors VI. BUDGET REQUESTED FOR THE FIRST YEAR Php 60, 000.00 VII. TOTAL BUDGET REQUESTED Php 120, 000.00 VIII. COOPERATING AGENCIES CHED, Selected DEPED Schools, DOST IX. OBJECTIVES The research project aims to:  Design and develop an equipment to facilitate the conduct and control the environmental factors that can affect the process and results of the brine shrimp lethality assay.  Test the effectivity of the equipment in maintaining its internal environment. 

Evaluate the usability and practicality of the product. 1

WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 2 of 10 Page No.

The project consists of four studies. Project: BRINE SHRIMP LETHALITY ASSAY SET – UP BENCH Study 1: Design and Development of a Brine Shrimp Lethality Assay Bench Specifically, this study aims to: 1. Produce a practical and user friendly equipment to facilitate the conduct of a Brine Shrimp Lethality Assay Study 2: Testing of the Effectivity of the Device in Maintaining the Internal Environment Specifically, this study aims to answer the question: 1. Is the device effective in maintaining the variables and uniformity of its internal environment particularly: a. Temperature b. Humidity c. Luminosity Study 3: Evaluation of the Usability and Practicality of the Product Specifically, this study aims to answer the question: 1. Is the product user friendly? 2. Is the product a practical investment for schools conducting cytotoxicity testing?

X. EXPECTED OUTPUT This series of studies is aimed to design and develop an equipment to facilitate the conduct of the brine shrimp lethality assay. One that will ensure maintenance and uniformity of the environmental factors to which the set – up of the brine shrimp lethality assay is exposed to. Furthermore, the effectivity of the device will be tested and its usability evaluated.

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WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 3 of 10 Page No.

XI. BACKGROUND/RATIONALE/SIGNIFICANCE Background The research requirement among junior and senior high school students is a good opportunity to apply their scientific skills. Providing them an avenue to explore and study in depth a subject they are interested in or something that is significant to their lives. But the problem of a significant majority of schools is the absence of equipment and materials they need to conduct the research activity not to mention their class laboratory activities (The National Academy & of Science and Technology, 2008), (Orbe, Espinosa, & Datukan, 2018). Brine shrimp lethality assay is a common, effective and simple method in assessing the toxicity of plant extracts (Meyer et al., 1982). It is extensively used as model organism for biochemical, physiological genetic and ecological studies (Browne, Wanigasekera, Wanigasekera, & G., 2000). According to Libralato, Prato, Migliore, Cicero, & Manfra, (2016), the main advantages of the process is its rapidity, cos-effectiveness and the readily available materials. Due to its simplicity one might think that it has been rendered obsolete by the more recent and high tech methodologies. But current studies have used the method including the testing of toxicity of silver nanoparticles (C. Arulvasu, S. M. Jennifer and D. Prabhu, 2013) and carbon-based nanoparticles (Mesarič et al., 2015). Study 1: Design and Development of a Brine Shrimp Lethality Assay Bench Rationale and Significance The researchers intend to develop an equipment that can facilitate the conduct of the brine shrimp lethality assay. A device that can control factors that can affect the result of the process, thereby provide a reliable data even though the venue of the experiment is less than ideal. Also the researchers have to think through its usability to allow students and teachers to use the product even with basic training only. Also its practicality as to be considered to enable even the small schools to acquire the product and make it available to as many students as possible. Study 2: Testing of the Effectivity of the Device in Maintaining the Internal Environment Rationale and Significance The effectiveness of the product is assessed to ensure that it can perform its expected function. The product will be designed to control the environmental factors that can affect the mortality rate of 3

WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 4 of 10 Page No.

Artemia spp. It is the test organism in brine shrimp lethality assay. Specifically, the capacity of the device to maintain the temperature, humidity and luminosity to a range that can be tolerated by the test organism have to be ensured. Study 3: Evaluation of the Usability and Practicality of the Product Rationale and Significance The persons who will have the last say on the usability and practicality of the product are the end users. The ease of use of the product is paramount because this will determine who could use the device. The study will also enable the makers to identify points of improvements that will make the device more user friendly and modify its design to suit the need of its user.

XII. REVIEW OF RELATED LITERATURE Toxicology. It is the science that deals with the study of the adverse effects of chemicals or physical agents on organisms (Lu, Yu, & Lu, 2018). Its level depends on factors like dose, duration of exposure and route of administration, shape and structure of the chemical itself and individual factors like age, gender and size (Samnani, 2011). The science of Toxicology is essential to evaluate the safety or hazards posed by certain substances (Erkekoglu, Giray, & Baaran, 2011). Studies related to it include carcinogenicity, developmental, reproductive toxicity, mutagenicity and neurotoxicity (Committee on Pesticides in the Diets of Infants and Children Board on Agriculture and Board on Environmental Studies and Toxicology & Council, 1993) The public policy statement of the Society of Toxicology states that “In the absence of human data research with experimental animals is the most reliable means of detecting important toxic properties of chemical substances and for estimating risks to human and environmental health” (Pope, Haldane, & Beeson, 2003). But the trend in the international legislation is moving towards the decrease in the use of vertebrates (Libralato et al., 2016) increasing the demand for standardized protocols using invertebrates or one that use computer simulation or in-silico testing (Bruinen de Bruin et al., 2009). Artemia is a genus of aquatic crustaceans also known as brine shrimp. Artemia, the only genus in the family Artemiidae (Asem, 2008). Annually, over 2000 metric tons of dry Artemia cysts are marketed worldwide for on-site hatching into 0.4 mm nauplii. The ability of the Artemia to produce dormant eggs, known as cysts, has led to extensive use of Artemia in aquaculture. The cysts may be 4

WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 5 of 10 Page No.

stored for long periods and hatched on demand to provide a convenient form of live feed for larval fish and crustaceans (Stappen, 2018). Artemia and Toxicology Study. The easy hatching from dry cysts and their year-round availability, Artemia is the most convenient test organism to toxicity study (Sorgeloos, Remiche-Van Der Wielen, & Persoone, 1978). In the review of toxicity testing protocols by Libralato et al., (2016) the 24 hour short-term and the 14 day long-term mortality are the most promising Artemia spp. protocols. Environmental Factors and Artemia spp. The study of (Naceur, Jenhani, & Romdhane, 2011) confirmed that factors like salinity, temperature, and dissolved oxygen for physicochemical parameters of water, as well as densities of total phytoplankton, diatoms, cryptophytes, and dinophytes, were the major factors affecting Artemia morphology. In addition, the study of Saygı & Demirkalp, (2002) the best growth and survival were obtained at 22-26°C, Artemia tolerated low temperatures very well. However, low temperatures caused a delay in development (e.g., Artemia reached 8.3 mm in 20 days at 30°C but required 30 days to reach the same length at 18°C). On the other hand, a rapid increase in mortality occurred at 30-32°C and no larvae in the 30°C treatment survived until day 30. XIII. WORK PLAN Methodology: Preliminary Activity The specification of the device will be listed together with the characteristics of its intended users. This is to ensure that the product will address the need to which it is designed and that it could be usable to its intended user. The different environmental factors that affect the survival of Artemia spp. will be noted. The researchers will identify several engineers and biologists and they will be requested to become consultant. There help will be requested to be able to come up with the most effective and efficient design to ensure that the environmental factors crucial to the survival of Artemia spp. are controlled and uniformly maintained. Aside from these other variables may be considered like the durability, convenience of use, presentable appearance and cost. These variables will be considered in the design and the choice of materials to be used. Procedure Design and Development of a Brine Shrimp Lethality Assay Bench 5

WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 6 of 10 Page No.

Based on the specifications and description gathered in the preliminary activity a drawing and a early cardboard prototype will be made. This will be used to initially assess ease of use of the product and if the materials untended to be used for the final prototype is feasible or some revision should be considered. Comments and suggestions from the consultants will be noted and evaluated to see if which could be practically incorporated into the product. The second plan of the device will be made and the working prototype will be constructed. The working prototype will be used for all the tests to assess the effectiveness, usability and practicality of the device. Testing of the Effectivity of the Device in Maintaining the Internal Environment After the working prototype is constructed, its capacity to uniformly maintain its internal temperature, humidity and luminosity in the different areas will be tested. Multiple points within the device will be identified to ensure that all areas where the set-up will be placed will have uniform environmental variables. The temperature, humidity and luminosity in the different location within the devise will be measured. This will be done with and without simulated set-up in the device. If no significant variation in the measured parameters (α-value at 0.05) is detected the device will be considered effective. Evaluation of the Usability and Practicality of the Product After the device has passed the initial testing the researchers will ask some teachers, students and technical experts to qualitatively evaluate the device. Some of them will be asked to do some simulated experiment utilizing the device, with the procedure and materials provided to them. Comments and suggestions will then be collected and evaluated to see if any refinement could be added into the product or whether a redesign is required.

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WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: WEST VISAYAS STATE UNIVERSITY URDC Issued by: Page 7 of 10 Page No.

CITED REFERENCES Asem, A. (2008). Historical record on brine shrimp Artemia more than one thousand years ago from Urmia Lake, Iran. Journal of Biological Research, 9, 113–114. Browne, R. A., Wanigasekera, G., Wanigasekera, , & G. (2000). Combined effects of salinity and temperature on survival and reproduction of five species of Artemia. Journal of Experimental Marine Biology and Ecology, 244(1), 44–29. https://doi.org/10.1016/S0022-0981(99)00125-2 Bruinen de Bruin, Y., Eskes, C., Langezaal, I., Coecke, S., Kinsner-Ovaskainen, A., & Hakkinen, P. J. (2009). Testing Methods and Toxicity Assessment (Including Alternatives). Information Resources in Toxicology, (0), 497–513. https://doi.org/10.1016/B978-0-12-373593-5.00060-4 Chinnasamy Arulvasu, Samou Michael Jennifer, Durai Prabhu, and D. C. (2013). The Scientific World Journal. The Scientific World Journal, 3(i), 342–347. https://doi.org/10.1126/science.279.5356.1431b Committee on Pesticides in the Diets of Infants and Children Board on Agriculture and Board on Environmental Studies and Toxicology, & Council, C. on L. S. N. R. (1993). Pesticides in the Diets of Infants and Children. Washington, D.C.: NATIONAL ACADEMY PRESS Washington, D.C. https://doi.org/10.17226/2126 Erkekoglu, P., Giray, B. K., & Baaran, N. (2011). 3R principle and alternative toxicity testing methods. Fabad Journal of Pharmaceutical Sciences, 36(2), 101–117. Libralato, G., Prato, E., Migliore, L., Cicero, A. M., & Manfra, L. (2016). A review of toxicity testing protocols and endpoints with Artemia spp. Ecological Indicators, 69, 35–49. https://doi.org/10.1016/j.ecolind.2016.04.017 Lu, Y., Yu, J., & Lu, Y. (2018). Artemia spp . Model - A Well-Established Method for Rapidly Assessing the Toxicity on an Environmental Perspective, 6(2), 1–15. Mesarič, T., Gambardella, C., Milivojević, T., Faimali, M., Drobne, D., Falugi, C., … Sepčić, K. (2015). High surface adsorption properties of carbon-based nanomaterials are responsible for mortality, swimming inhibition, and biochemical responses in Artemia salina larvae. Aquatic Toxicology, 163, 121–129. https://doi.org/10.1016/j.aquatox.2015.03.014 Meyer, B., Ferrigni, N., Putnam, J., Jacobsen, L., Nichols, D., & McLaughlin, J. (1982). Brine Shrimp: A Convenient General Bioassay for Active Plant Constituents. Planta Medica, 45(05), 31–34. https://doi.org/10.1055/s-2007-971236 Naceur, H. Ben, Jenhani, A. B. R., & Romdhane, M. S. (2011). Influence of environmental factors on the life cycle and morphology of Artemia salina (Crustacea: Anostraca) in Sabkhet El Adhibet (SE Tunisia). Biological Letters, 48(1), 67–83. https://doi.org/10.2478/v10120-011-0008-6 7

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Orbe, J. R., Espinosa, A. A., & Datukan, J. T. (2018). Teaching Chemistry in a Spiral Progression Approach : Lessons from Science Teachers in the. Australian Journal of Teacher Education, 43(4). https://doi.org//10.14221/ajte.2018v43n4.2 Pope, A., Haldane, J. B. S., & Beeson, P. (2003). Animals in research, 316–340. Samnani, A. (2011). International Journal of Biomedical and Advance Research, 02. Saygı, Y. B., & Demirkalp, F. Y. (2002). EFFECTS OF TEMPERATURE ON SURVIVAL AND GROWTH OF ARTEMIA FROM TUZ LAKE, TURKEY_Yasemin Basbug Saygı_2002.pdf, 54(3), 125–133. Sorgeloos, P., Remiche-Van Der Wielen, C., & Persoone, G. (1978). The use of Artemia nauplii for toxicity tests-A critical analysis. Ecotoxicology and Environmental Safety, 2(3–4), 249–255. https://doi.org/10.1016/S0147-6513(78)80003-7 Stappen, G. Van. (2018). 4 . 1 . Introduction , biology and ecology of Artemia. Retrieved August 26, 2018, from http://www.fao.org/docrep/003/w3732e/w3732e0m.htm The National Academy, & of Science and Technology. (2008). BIOLOGY EDUCATION : GAPS , ISSUES , AND CONCERNS. Retrieved August 25, 2018, from http://www.nast.ph/images/pdf files/Publications/Monograph Series/NAST Monogrpah Series 16.pdf

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XIV. Schedule of Activities Date

Activities

Preliminary Activity Brainstorming for the June 1, – product specification and Aug.15, 2019 for the characteristics of the users Sept. 1 – Design, initial prototype December 30, construction and its initial 2019 evaluation Finalization of the plan Jan. 1 – March and construction of the 30, 2020 working prototype Testing for the Mar. 30, – June effectiveness of the 30, 2020 working prototype Adjustment of the working prototype and July 1, 2010 to conduct of the usability Jan 30, 2021 and practicality evaluation. Feb. 1 – June Write up 30, 2021

Expected Work Output (%)

Amount

5%

Php 20,000.00

15%

Php 50,000.00

45%

Php 135,000.00

60%

Php 15,000.00

80%

Php 35,000.00

100%

Php 10,000.00

XV. SUMMARY OF ANNUAL FINANCIAL PLAN For July 1, 2018 – March 30, 2019 PARTICULARS I. II.

Personal Services Research Assistant Maintenance and operating expenses Local Travel Expenses Communications

BUDGETARY REQUIREMENT 50,000.00

25,000.00 9

WVSU-RDC-SOI-01-F01 Document No. WVSU RESEARCH PROPOSAL FORMAT 1 Issue No. 0 Revision No. April 27, 2018 Date of Effectivity: URDC Issued by: WEST VISAYAS STATE UNIVERSITY Page 10 of Page No. 10

Supplies & materials Professional Services: Honorarium for the panel of consultants TOTAL COST For June 1 2010-June, 2021 I. Personal Services Incentive Claim of Researchers Research Assistant Statistician II. IP processing Expenses III. Maintenance and operating expenses Local Travel Expenses Printing and Binding Expenses Professional Services:

10,000.00 1,000.00 85, 000.00

P171, 000.00

P 50,000.00 48,000.00 500.00 P 50,000.00 P

10,000 2,500.00 5,000.00

III. Equipment TOTAL COST GRAND TOTAL

P166,000.00 P337,000.00

Prepared by:

PRENCY D. YERRO

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