The Use of Ultrasonic Waves as an Alternative Method in Mitigating Algal Blooms

By Madrid, Ludhovik Luiz B. Medrana, Micholo Lanz B. Morales, Justin Marius D.

Submitted to the Faculty of the Philippine Science High School Main Campus in partial fulfillment of the requirements for Science and Technology Research 1 March 2011

ABSTRACT
One type of Harmful algal bloom (HAB) may cause the discoloration of seawater caused by a dense population of dinoflagellates. However, not all types of HABs can cause water discoloration. HABs have been recurring in Philippine waters since 1988 and are notable for their disruption to the marine ecosystem and their negative health and economic effects. The aim of this project is to determine the effects of time exposure of ultrasound waves on Pyrodinium bahamense var. compressum to know whether these variables would lead to inhibition of growth or complete elimination of the organisms. From an initial culture of the dinoflagellate P. bahamense, 24 Erlenmeyer flasks containing a 100 mL culture solution will be prepared and divided into 12 separate experiment groups of 2 flasks each. Each experiment group will test the effect of a same frequency (1 MHz) on a sample exposed for different time spans. Control samples will receive no treatment. After each treatment is performed, a cell count will be taken of the sample using a Sedgewick-Rafter slide and recorded. The results of each experiment group will be statistically analyzed to determine any significance between the treatments and the control. This analysis will help determine the optimum exposure time for inhibition of growth or complete elimination of the organism.

APPROVAL SHEET
This research work entitled, The Use of Ultrasonic Waves as an Alternative Method in Mitigating Algal Blooms by Madrid, Ludhovik Luiz B., Medrana, Micholo Lanz B., and Morales, Justin Marius D., presented to the Faculty of the Philippine Science High School Main Campus in partial fulfillment of the requirements in Science & Technology Research 1, is hereby accepted. ____________________________________ Dr. Jessamyn Marie O. Yazon, Ph.D. Research Adviser

ACKNOWLEDGMENTS
First, we would like to thank ourselves, for doing our best in working on this research, and in winning the oral defense in YMSAT. We would like to thank our classmates, for coming along with us while we cram our STR requirements. We would like to thank our research teacher, Maam Yazon, for reviewing on our work, and pointing out mistakes and improvement on our STR requirements. We would also like to thank some of our teachers: Maam Chupungco, Maam Docto, Maam Buenafe, and Sir Tan for giving us advice during the oral defense and Sir Talaue for giving us the research journals that we cannot access. We would also like to thank the people of MSI (Sir Garry, Ate Jenelle, Maam Lita, Dr. Azanza) for helping us in our research, Williard Jose (III-Be), Dr. Jose, and Dr. Escoto, who helped us find a contact person who has an ultrasound machine, and Mr. Publico, for letting us borrow his ultrasound cleaner. Last but not the least; we would like to thank God, for giving us the wisdom, strength, and inspiration to finish our research, and for giving us hope whenever we had problems in STR.

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TABLE OF CONTENTS
Page Approval Sheet Acknowledgments Table of Contents List of Tables List of Figures I. Introduction A. Background of the Study B. Statement of the Problem C. Significance of the Study D. Scope and Limitations II. Review of Related Literature A. Harmful Algal Bloom (HAB) B. Algal Culture C. Ultrasonic Principles III. Materials and Methods A. Procurement of Algal Solutions and Other Materials B. Preparation and Sterilization of Culture Flasks C. Preparation of Algal Solutions D. Initial Cell Counting E. Setting up of Ultrasonic Cleaner 14 14 15 19 20 5 9 10 1 2 2 3 i ii iii v vi

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F. Exposure of Algae to Ultrasound and Final Cell Counting G. Computation, Graphing, and Tabulation of Collected Data H. Statistical Tests (ANOVA and T-test) IV. Appendices A. Summary of Materials and Methods B. Formulas, Tables, and Graphs needed for Data and Analysis C. Risk Assessment D. Task List E. Materials Sourcing and Budgeting F. Gantt Chart G. Network Chart Bibliography

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26 27 29 32 34 36 36

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LIST OF TABLES
Table 5.2.1 5.2.2 5.2.3 5.2.4 Title Table for Analysis of Variance (ANOVA) Table Comparing Initial and Final Cell Density for each Treatment Table for Correlated T-Test Initial, Final, and Change of Cell Densities of Pyrodinium bahamense var. compressum cultures in Concentration of Algae vs. Time of Exposure 5.3 5.4 5.5.1 5.5.2 Risk Assessment Table Task List of the Methods in the Research Study Table of Materials needed for Experiment and their Costs Table of Transportation and Electricity Consumption 29 32 34 34 Page 27 27 27 28

LIST OF FIGURES
Figure 3.2.1 Title Erlenmeyer flasks obtained in MSI (left picture), and Erlenmeyer flasks were cotton seal (right picture) 3.2.2 Water filterer connected to a container for filtered seawater (left picture). Erlenmeyer flask filled with filtered seawater (middle picture). Inside the autoclave are Erlenmeyer flasks with filtered seawater and seals covered with aluminum (right picture) 3.3.1 Things needed for preparation of algal solutions. From left to right: stock culture, alcohol lamp, single channel pipette, autoclaved Erlenmeyer flasks with filtered seawater, and F/2 medium. 3.3.2 Aseptic methods for sterilization purposes. Erlenmeyer flasks mouth was heated (left), and F/2 containers mouth was also heated (right) 3.3.3 3.3.4 Transferring of F/2 medium from container to Erlenmeyer flask Transferring stock culture to Erlenmeyer flask near the open flame (aseptic method) 3.3.5 3.4.1 All 24 flaks kept near a light source Things needed for cell counting (from left to right): Eppendorf tubes in orange case, culture flask, and single channel pipette. 3.4.2. 3.5. 5.1. Researchers working on cell count while looking in the microscope Ultrasonic Cleaner Set-up Process Flowchart for The Research Study 20 20 26 18 19 17 17 17 16 15 Page 14

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5.6 5.7

Gantt Chart with Expected Dates of Work Network Chart of the Research Study

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I. INTRODUCTION
A. Background of the Study A Harmful Algal Blooms (HAB) is a discoloration of seawater caused by a dense population of a given species of dinoflagellate (Villao, 1988). In some cases, blooms are accompanied by toxins which, when ingested by surrounding marine life, are capable of poisoning them or their predators. In the Philippines, especially in the waters of Manila Bay, HABs are caused by the dinoflagellate Pyrodinium bahamense var. compressum, which have recurred here since 1988 (Azanza, 1997). HABs have a direct effect on the coast and its underlying areas. Examples of such effects include impacts like fishkill (which is caused either by poisoning or oxygen depletion) and Paralytic Shellfish Poisoning (PSP), a disease in humans caused by the consumption of tainted shellfish resulting in either partial or complete paralysis of the body (Villao, 1988). It is these negative effects of HABs that have motivated scientists in multiple related fields to devote their research efforts on finding methods to reduce HABs.

One method that has not been attempted yet (as far as the group's preliminary research went) is the use of ultrasound to control HABs. Ultrasound has proven successful as a method of controlling freshwater algal blooms (caused by cyanobacteria) in agricultural areas, both locally and overseas (Song, et al., 2005). Removal of cyanobacteria in these environments is needed due to their oxygen depleting effects with regards to agriculture and their toxic nature, especially in sources of potable water (Falconer, 1999).

The use of ultrasound to control freshwater algal blooms employs the concept of microscopic cavitation resulting from high frequencies, resulting in biological disintegration. Stemming from this concept, the research will see whether the use of these ultrasound waves can be applied in the control of HAB's, bringing us to the statement of the problem.

B. Statement of the Problem This study aims to test whether the exposure of harmful algal blooms (HABs), specifically those caused by the blooms of the dinoflagellate Pyrodinium bahamense var. compressum, to ultrasound waves would result in the mitigation or reduction of these blooms. Assuming this is feasible, the secondary objective of this study is to determine the ideal length of time it would take for ultrasound exposure to effectively control HABs. The dinoflagellates population size and concentration are aspects that will be observed and measured during the course of this study. This study also attempts to see whether the ultrasound transducer to be used in the experiment can easily be deployed on the field, without necessarily experimenting outside of a controlled laboratory setup. Accomplishing these goals will lead to the real life problem, which is to find a method of controlling HABs.

C. Significance of the Study In the research of HAB's, the field of studying Harmful Algal Bloom control has been progressing far too slowly. Though there are new methods of harmful algal bloom control (use of chemicals, genetically engineered species, clay flocculation), these methods have harmful effects on the environment and biological life other than HAB's (Anderson, 2005).

With the incremental develop of the industry and economy, the HAB (Harmful Algal Bloom) problems gradually become a cosmopolitan marine disaster, which endangers the health of the people and the fishery ecosystem (Jinhui, 2005). Fisheries in the Philippines suffer from fish kills caused by these HABs, and the lives of seafaring Filipinos are also more at risk. Using the methods for HAB control that were mentioned above would only worsen the problem, highlighting the need for a practical method that will not bring harm to species other than HABs. This problem is solved by the use of ultrasound to control populations of HABs, which would also reduce the number of fish kills. This would in turn cause an increase of the fish supply of our country and decrease the number of fatalities caused by PSP. Thus, solving this problem would help our countrys poverty problems and its economic status. D. Scope and Limitations The study will be done over a course of around one and a half months. Cultures of Pyrodinium bahamense var. compressum from the Marine Science Institute (MSI) wasused in the research study. Twelve groups consisting of two replicates each will be exposed to different lengths of time. The frequency of ultrasound that was used is 1 MHz. The time of exposure (independent variable) will vary from each treatment, ranging from 1 hour to 6 hours. The experiment will find out if the optical density, the dependent variable, will increase, decrease, or remained unchanged. Other than the frequency of the ultrasound, time of exposure to light, volume of solution, amount of nutrients, salinity, and temperature will be considered as controlled variables. This study aims to cover the control of the population of HABs and does not deal with the complete elimination of HABs.

This study is completely different from the study entitled Using Ultrasound to Control Algal Blooms which was conducted by Dr. Carl Howard and a team of researchers from the University of Adelaide. The research of the aforementioned team deals with the effects of ultrasound to blooms of blue-green algae, whereas this research tests if various time exposures to ultrasound have an effect on HABs.

II. REVIEW OF RELATED LITERATURE

A. Harmful Algal Bloom (HAB) What is a HAB? An HAB, commonly known as red tide, is the discoloration of a body of water caused by massive populations of coastal dinoflagellates (Villao, 1988). Commonly distributed in the tropical Indo-Pacific region, HABs are predominant in numerous countries including the Philippines (Busine, et al., 2003). Factors affecting HAB growth Dinoflagellates within HABs prefer high surface-water temperatures and high light intensities, though this does not mean that HABs only occur in tropical areas. HABs occur in hot, calm weather because surface temperatures warm up even in normally cool areas (Badylak, et al., 2004; Maclean, 1977). Light wind intensity moves the bloom near the coast, whereas strong wind intensity aids in the swimming of dinoflagellates to the surface. Storms on the other hand, even with strong wind intensity, disperse the HABs. Storms also result in the death of dinoflagellates and can prevent the development of red tides (Pollingher & Zenel, 1981). Red tides usually occur after an upwelling has stopped, but the nutrients brought to the surface do not appear to be the direct cause of these blooms (Grindley & Nel, 1970). The overloading of nutrients to bodies of water, or eutrophication, gives favorable conditions to HABs. The presence of HABs, however, has proved to be detrimental to other organisms in the environment (Nybakken, 1982). Effects of HAB to marine and human environment

HABs have been a significant global and national concern due to their negative public health and or/economic effects (Fernandez & Ricafrente, 2010). Red tides deplete the nutrients needed for other marine organisms to survive. They also cause oligotrophication, which is the abundance of toxic materials in bodies of water. HABs have the ability to produce very lethal toxins like saxitoxin, Neo-saxitoxin and decarbamoyl, which can cause the death of marine organisms. These toxins can also accumulate in some animals, and can result in Paralytic Shellfish Poisoning (PSP) in humans if these animals are eaten. Symptoms of PSP include paralysis, vomiting, shortness of breath and other difficulties, and can result in death through respiratory failure (Busine, et al., 2003). HABs have economic effects on multiple sectors of society. Fish kills caused by HABs reduce fish supply, while shellfish bans imposed by local authorities caused losses in the fishing industry, pose problems to international trade, and result in underemployment of industries (Bajarias, et al., 2003).

Methods of HAB management and monitoring in the Philippines Current methods of HAB management are limited to detection and analysis of the areas affected by a bloom, including the implementation of bans on shellfish and similar products affected, and an information campaign by various government agencies. This management method has been done since 1984, and is inefficient in the sense that very little is done to control the bloom physically, and simply involves waiting for it to fade out (Bajarias, et al., 2006). Other physical means, such as domoic acid treatment, of controlling and eliminating HABs are still being tested out by various government agencies such as the PCAMRD (Fernandez & Ricafrente, 2010).

However, the Philippine government keeps on monitoring seas that are known to have past cases of red tide. The Department of Health (DOH), and Bureau of Food and Drugs (BFAD) keeps on acquiring samples of seawater from the seawater to obtain quantitative analysis of cell density and toxin concentration. Aerial surveillance is also monitored by the Department of Agriculture (DOA), and Philippine Air Force, to see if there is any discoloration of the seas surface (Busine, et al., 2003). Methods of HAB management and monitoring outside Philippines Countries have advanced technologies that the Philippine lacks, and one of them is the satellite monitoring system (Bajarias et al., 2003). Countries such as China, Norway, Canada, Japan, and USA use remote sensing satellites to detect HABs. With the use of satellites, aerial surveillance is much easier (Andersen, et al., 2001; Busine, et al., 2003). They also train fishermen on how to detect HABs and collect HAB samples with the use of buoys, lighthouses, and plankton nets. Also, the marine farms are more advanced than in the Philippines; their fish and mussel farms contain laboratories that separate the poisoned marine organisms from the healthy ones (Anderson, et al., 2001; Bajarias, et al., 2003). These countries also developed ways on how to control the population of algae, but these have limitations: adding of chemicals, flocculation, and biological manipulation. Adding of chemicals started when the US government added copper sulfate to seas with HABs near Florida. Adding copper sulfate was effective, but after weeks, the HAB reestablished itself, killing more fish along the shores of Florida, and calm wind currents also carried part of the bloom to other seas of United States. They also used other chemicals such as ozone and aponin, but these have negative effects in marine life other than harmful algae. Flocculants, substances that capture suspend particles until they become heavy and fall as

sediments, were tested in Japan and were found to be effective against HABs, but it is very expensive. Japanese researchers also used the addition of viruses to destroy the algae, and bacteria, dinoflagellates, and other zooplankton to compete with the algae. It was effective, but it also massive bivalve and fish kills (Andersen, et al., 2003; Newcombe, 2009). Pyrodinium bahamense var. compressum In the Philippines, Pyrodinium bahamense var. compressum has been identified as the main organism responsible for HAB outbreaks in Manila Bay since 1988. P. bahamense forms its blooms by cell division during their vegetative stage, and is easily grown in a laboratory setup, given the proper conditions. P. bahamense has a growth rate of one cell division every three days, and produces a planozygote in later stages of its bloom because of a union of its gametes. A non-motile hypnozygote is then formed, which brings the organism into dormancy for about three to four months, unless optimal conditions of salinity, temperature, and light intensity are established. Once these conditions are met, the hypnozygotes then germinate via meiosis and then binary fission, in turn initiating a new bloom (Azanza, 1997). Blooms of P. bahamense are dominant in Bolinao, Masinloc, Manila, Palawan, Camiguin, Surigao Province, Leyte, and Samar. These areas are reportedly to have massive fish kills, especially in the Manila Bay. However, the effects of El Nino and La Nina disrupt the normal growth pattern of the algae, so fish kills in these areas do not happen at the same time (Azanza, 1997; Bajarias, et al., 2003; Busine, et al., 2003). Saxitoxins are considered the most potent neurotoxin found in dinoflagellate blooms (Busine, et. al., 2003). Aside from Saxitoxin, Pyrodinium also produces other types of neurotoxins, particularly Neo-saxitoxin, decarbamoyl, gonyautoxin-5 and gonyautoxin-6

(Busine, et. al., 2003; Corrales, 1991). With these toxins, it is necessary to note that proper algae culturing protocols would need to be implemented in this research, for the safety of the researcher (Azanza, 1997). B. Algal Culture Factors considered in growing algae Like all organisms, certain factors should be considered in growing algae. As with all plants, algae must have sufficient nutrients to support growth, and factors, such as light, temperature, salinity, seawater quality, mixing and cleanliness will all need to be kept constant to ensure favorable conditions for algal growth (Food and Agriculture Organization of the United Nations, 2007; Hallegraeff et al., 1995). Techniques in growing algae Aseptic method is strictly imposed when culturing microorganisms. It prevents addition of unwanted microorganisms. While adding nutrients, or the stock culture, you must make sure you heat the mouth of the container, or transfer the necessary components near an open flame (Food and Agriculture Organization of the United Nations, 2007). In an algal culture system, a stock culture should be maintained. These stock cultures provide the reservoir of algal cells from which to start the larger-scale cultures used for feeding. In preparing the replicate culture flasks, use the pipette to transfer a small sample of the stock culture to the flasks. Use a separate pipette for transferring the nutrients. For dinoflagellates, their nutrient is F/2 medium, a mixture of vitamins, nitrates, phosphates, silicates, and other minerals (Food and Agriculture Organization of the United Nations, 2007).

Algal cultures must be monitored daily to detect if there are dead species, cell aggregation or clumping, or contaminants. If a culture contains multiple clumped cells, cells with cell walls broken, more than one species present, or is contaminated with foreign bodies, the culture should be discarded (Food and Agriculture Organization of the United Nations, 2007). Cell counting methods Monitoring of the algal cultures will also involve a cell count, using either a haemocytometer or a Sedgewick-Rafter counting slide. A haemocytometer is widely used by researches because of its easy instructions, its lightweight use. However, haemocytometer is not suitable for algae larger than 45 microns, such as P. bahamense. Haemocytometers also rely on calculations and estimation, which can be inaccurate (Karlson, et al., 2010; Tech Note, 2004). This research study proposes the Sedgewick-Rafter counting slide, a traditional counting method. It contains a 20 by 50 grid of 1 mm2 squares. It is recommended with cells of large size and population. It does not need calculations, but the counting is manual and time-consuming (Karlson et al., 2010). Aside from establishing principles and culturing techniques of HABs, this research will also note the scientific concept of ultrasound and its effects on biological and chemical materials. C. Ultrasonic Principles Basic Ultrasonic Principles Ultrasound is defined as a high frequency that is above 20 kHz, above the audible range. Ultrasound waves require an elastic medium, such as solids or liquids, for

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transmission, operated by stressing the medium itself. Its wavelengths are small compared to audible waves, and it is said to have a high frequency relative to the audible range of humans (Agarwal, 2005). The most common methods of ultrasonic examination utilize either longitudinal waves or shear waves, and other forms of sound propagation exist, including surface waves and Lamb waves. Surface waves are characterized by elliptical particle motions and are bound to the surface of a material, while Lamb waves are characterized by complex vibrations in materials where its thickness is less than the ultrasound wave induced to it (Olympus, 2006). Ultrasound can be classified into two types: low frequency ultrasound, and high frequency ultrasound. Low frequency ultrasound ranges from 20 kHz to 1 MHz, and high frequency ultrasound ranges from 1 MHz onwards (Van Iersel, 2008). Biological Effects of Ultrasound There are ways in which ultrasound can produce biological effects such as cavitation, microstreaming, and heating (Chudleigh & Thilaganathan, 2004). Cavitation is the ultrasonically induced activity occurring in a liquid or liquid-like material that contains bubbles or pockets containing gas or vapor (OBrien, 2007), and can rupture the cell membranes of microorganisms (Li, 2009). Microstreaming is the formation of small local fluid circulations, and can be intra- or extracellular. Microstreaming can cause the same effects to those of cavitation. Heating is caused by the absorption of the ultrasound wave by organisms, and it can cause internal injuries to the cell (Chudleigh & Thilaganathan, 2004). Low frequency ultrasound is enough to produce cavitation, microstreaming, and heating in aquatic environments (OBrien, 2007; Van Iersel, 2008).

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In tissues of macroscopic animals, high frequency ultrasound can only cause abrasions in the tissues. Heating and not cavitation is the cause of tissue damage in animals. As frequency increases, absorption of ultrasound waves increases. As the tissue absorbs the ultrasound waves, their vibration causes to increase the internal energy of the tissue, which creates heat (Carstensen, et al., 1974). Chemical Effects of Ultrasound In cavitation, the induced activity of bubbles or pockets can grind insoluble substances in liquid. These insoluble substances will become smaller until it suspends in the liquid medium (Li, 2009). Ultrasound can also decompose or transform organic molecules, such as chlorophyll and saxitoxin, into smaller and useless ones. Ultrasound increases the energy needed to break the bonds between atoms, until the energy reaches the activation energy, the minimum energy required to have a chemical reaction. Once the organic molecule changed its form, it can no longer function properly (Emery, et al., 2005; Van Iersel, 2008). If the harmful algae lacked the necessary compounds needed for photosynthesis, it can no longer survive (Andersen, et al., 2003). C. Transducer, the Ultrasound Machine What is the transducer? The device that both generates the ultrasound and detects the returning echoes is the transducer. Transducers are made of materials that exhibit piezoelectricity, which is electric polarity due to pressure especially in a crystalline substance (Fleischer, et al., 1991). In ultrasonics, the piezoelectric material is the actual transducer because it converts ultrasound into electric energy and vice-versa. When voltage is applied, the transducer will expand and contract, creating ultrasound (Chudleigh & Thilaganathan, 2004; Sherman & Butler, 2007).

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Main Components of Transducers There are three main components of transducers, namely the active element, the backing, and the wear plate. The active element is the piezoelectric material. The backing acts as an energy conserver by absorbing the energy radiating from the active element. Finally, the wear plate protects the transducer element from the surroundings (Fleischer, et al., 1991; Olympus, 2006). Sonicators A sonicator is a device used to break open cells using ultrasound waves, a method called sonication. Sonicators make use of the properties of high frequency ultrasound to disrupt the cell wall of an organism. Sonication is employed in most studies for extraction of enzymes within a cell, with insoluble materials separated by centrifugation (Madison Area Technical College, 2005).

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III. MATERIALS AND METHODS

A. Procurement of Algal Solutions and other materials A stock culture of Pyrodinium bahamense var. compressum was obtained from the Marine Science Institute (MSI) at the University of the Philippines (UP) Diliman Campus. An ultrasonic cleaner was obtained from the I-MAT Pro Company. The SedgewickRafter counting slide, materials for the construction of the culture tank and all other laboratory equipment (stirring rod, pipette, waste bottles, Erlenmeyer flasks, reagent bottles, F/2 media, microscope, autoclave, Lugols Iodine, Eppendorf Tubes, etc.) was obtained from the MSI. Common materials for cleaning (at the end of the experiment) were bought from public markets. B. Preparation and Sterilization of Culture Flasks Twenty-four (24) 125 mL Erlenmeyer flasks were used in the experiment. A seal for the flasks were made by rolling a thick sheet of cotton. The roll of cotton was inserted in the hole of the flask to check if it fits. Otherwise, a portion of cotton must be removed. The process must be repeated several times until the cotton tightly fits in the hole. Then, the cotton was wrapped with cheesecloth and twisted to form a handle in the top. The handle was sealed with masking tape.

Figure 3.2.1. Erlenmeyer flasks obtained in MSI (left picture), and Erlenmeyer flasks were cotton seal (right picture).

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32 ppt of seawater was filtered by using a water filterer. Seawater must be filtered to remove unwanted substances (other than salts) (Karlson, et.al, 2010). The filtered seawater was transferred in a large flask, and each Erlenmeyer flask was filled with 50 mL of filtered seawater. Then, the seals were covered with aluminum foil.

Figure 3.2.2 Water filterer connected to a container for filtered seawater (left picture). Erlenmeyer flask filled with filtered seawater (middle picture). Inside the autoclave are Erlenmeyer flasks with filtered seawater and seals covered with aluminum (right picture). Autoclaving is necessary to remove unwanted microorganisms inside and outside of the flask (FAO, 2007). All flasks were placed inside an autoclave for 1 hour and after autoclaving; they were cooled for the day. C. Preparation of Algal Solutions The preparation of algal solutions was executed in a secure, temperature-controlled room. All stock cultures must be kept in a temperature-controlled room, to control (not alter) the growth pattern of the algae (Food and Agriculture Organization of the United Nations, 2007; Karlson, et.al, 2010). Aseptic method was used to prevent contamination of unwanted organisms in the flasks (Food and Agriculture Organization of the United Nations, 2007).

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Figure 3.3.1 Things needed for preparation of algal solutions. From left to right: stock culture, alcohol lamp, single channel pipette, autoclaved Erlenmeyer flasks with filtered seawater, and F/2 medium. F/2 medium must be added to each flask before adding the algae. F/2 medium is a broth for the algaes nutrients. Without F/2, the algae will not survive (Food and Agriculture Organization of the United Nations, 2007). Before adding the F/2, each mouth of the F/2 container and the flask was heated by an alcohol lamp (aseptic methods). A ratio of 1 L of algal solution to 2 mL of F/2 was used, since this is the protocol MSI imposed on growing P. bahamense var. compressum. Each flask contained 100 mL of algal solution, so 0.2 mL, or 200 microliters of F/2 was added, by using a single channel pipette. After each flask has been added with F/2 medium, the mouth of the stock container and the flask was heated, and 50 mL of P. bahamense var. compressum was added, until all 24 flasks were filled. After this, all flasks were randomized and labeled acc. to their assigned treatment. There were 1 control and 11 treatments, and each group has two flasks.

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Figure 3.3.2. Aseptic methods for sterilization purposes. Erlenmeyer flasks mouth was heated (left), and F/2 containers mouth was heated (right)

Figure 3.3.3. Transferring of F/2 medium from container to Erlenmeyer flask.

Figure 3.3.4. Transferring stock culture to Erlenmeyer flask near the open flame (aseptic method).

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The algae will die if there is no light because of absence of photosynthesis (Hallegraeff et al., 1995). All flasks are kept in the corner of the room near a light source.

Salinity, temperature, light intensity, and wind intensity are the factors to be considered in the habitat of P. bahamense (Hallegraeff et al., 1995). Salinity was kept constant by adding the same amount of seawater (in the same concentration of 32 ppt). Since the flasks are kept in a temperature-controlled room, controlling temperature is not much of problem. The algae are inside the flasks, which does not have air disturbance. Light intensity was kept constant by the MSI staff monitoring the time of using the light source in the room.

Figure 3.3.5. All 24 flaks kept near a light source.

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D. Initial Cell Counting

Figure 3.4.1. Things needed for cell counting (from left to right): Eppendorf tubes in orange case, culture flask, and single channel pipette. The number of cells in the cleaner tank was counted by using a 20 x 50 SedgewickRafter counting slide before exposing them in ultrasound waves. A sample of algal solution was be obtained by using a single channel pipette, and it must be preserved in Lugols Iodine before counting (Karlson, et al., 2010). 1 mL of the solution was obtained at it was transferred in an Eppendorf tube. Two 1 mL samples were obtained for each flask, because the counting consists of two trials. Then, each tube was added with 1 drop of Lugols Iodine by using a dropper. Adding the Lugols Iodine was done outside the room since its fumes can kill the algae (Karlson, et al., 2010). Then, the 1 mL solution was transferred, by using a different dropper, to the cover glass of the counting slide. The cover glass must be slowly swung so it completely covers the solution. Careful alignment of the cover glass will stop air bubbles from proliferating into the sample and will ensure that the solution is completely spread in the cover glass. If the solution is completely spread, the slide is ready for counting.

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Figure 3.4.2. Researchers working on cell count while looking in the microscope. The cover glass of the counting slide was viewed in a microscope using a 10x objective. A counter was used to aid the researcher in counting cells. While counting, if the researcher sees x cells, he will click the counter x times. All 1000 squares were checked and the number of cells was recorded. For faster counting, the squares in the slide were scanned in a zigzag formation (Karlson et al., 2010). E. Setting up of ultrasonic cleaner

Figure 3.5. Ultrasonic Cleaner set-up.

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The ultrasonic cleaner served as the culture tank of the experiment, and it was filled with tap water. Sound waves can pass through solids and liquids, therefore algae can still be exposed through ultrasound waves (Olympus, 2006). Pyrodinium bahamense var. compressum can survive in subtle amount of air (Azanza, 1995), so there is no need of oxygen/carbon dioxide tanks. 2 flasks were placed inside the ultrasonic cleaner. Only the inside of the cleaner must be wet, and the level of the water must not rise beyond the mark inside the cleaner, to prevent electrocution (Lee, 2004). If the level of the water goes up beyond the mark, the flasks were discarded and removed some of the water, until the water level is exactly at the mark. F. Exposure of Algae to Ultrasound, and Final Cell Counting After setting up the ultrasonic cleaner, it was switched on. Each treatment was applied with the same amount of frequency (1 MHz), and with varying amount of time. The first treatment was exposed with 1 hour of ultrasound. Each succeeding treatment was exposed with an additional 30 minutes of exposure (90 minutes for the 2nd treatment, 120 minutes for the 3rd, and so on). A countdown timer was used to monitor the time of exposure for each treatment. At the end of each treatment, the flasks were taken out and the same process (two trials) was done for counting the algae. After finishing all treatments, the water inside the cleaner was removed. The cleaner was wiped with tissue until its dry, and it was kept back in its container. G. Computation, Graphing, and Tabulation of Collected Data All data computed or obtained before were presented into tables and graphs using Microsoft Excel. There will be 12 tables, and each table is assigned for one treatment. The

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table will consist of the initial cell density and final cell density of the treatment. Then, each table will be interpreted by using line graphs. H. Statistical Tests (ANOVA and T-Test) Analysis of Variance (ANOVA) will be used, with a level of significance of 0.05, to find out if there is a significant difference between the change of cell densities of each treatment (alternative hypothesis), or if there is no significant difference (null hypothesis). A t-test will be used, for each treatment with a level of significance of 0.05, to find out if there is a significant difference between the initial and final cell densities of the treatment (alternative hypothesis), or if there is no significant difference (null hypothesis).

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IV. BIBLIOGRAPHY
Agarwal, S. K. (2005). Advanced biophysics. New Delhi: APH Publishing. (Agarwal, 2005) Anderson, M., Andersen, P., Bricelj, V.M., Cullen, J., & Rensel, J. (2003). Monitoring and management strategies for harmful algal blooms in coastal waters. Paris: UNESCO. Azanza, M.P., Azanza, R.V., & Ventura, S. (2003). Varied assays for PSP toxins in heatshocked Philippine green mussels (Perna viridis). Journal of food safety, 23, 249259. Azanza, R.V. (1997). Contributions to the understanding of the bloom dynamics of P. bahamense. Science Diliman, 9(1-2), 1-6. Azanza, R.V., & Hall, S. (1993). Isolation and culture of Pyrodinium bahamense var. compressum from the Philippines. USA: Elsevier. Azanza, R.V., Cruz, L.J., Carino, F.A., Blanca, A.G. & Butardo, V.M. (2009). Paralytic shellfish toxin concentration and cell density changes in Pyrodinium bahamense Noctiluca scintillans feeding experiments. Toxicon, 3(109) Azanza, R.V., Dela Rosa, A., Sombrito, E.Z., Cruz, L., Siringan, F.P., McGlone, M.S.D., & Duyanen, J. (2001). Harmful algal bloom (HAB) management lessons from multidisciplinary research program in Manila Bay, Philippines. Philippines: DOST. Badylak, S., Kelly, K., & Philips, E. (2004). A description of Pyrodinium bahamense (Dinophyceae) from the Indian River Lagoon, Florida, USA. Phycologia, 43(6), 1317. Bajaras, F., Relox Jr., J., & Fukuyo, Y. (2006). PSP in the Philippines: three decades of monitoring a disaster. Coastal Marine Science, 30(1), 104-106. Busine, M.B., Cardenas, J., Khonghun, G., Pelobello, M.R., Raymundo, E., & Reyes, C. C. (2002). The killer tide: The impacts and monitoring of red tide. Ekolohiya, 1(1), 1-8. Carstensen, E.L., Miller, M.W., & Linke, C.A. (1974). Biological effects of ultrasound. Journal of Physical Biological Sciences, 2, 173-192. Cell counting and dye exclusion viability assays using haemocytometer. Tech Note (2004), 3(25), 1-2. Chudleigh, T., & Thilaganathan, B. (2004). Obstetric ultrasound (3rd Ed.). Philadelphia, USA: Elsevier Limited. Emery, R.J., Papadaki, M., Freitas dos Santos, L.M., & Mantzavinos, D. (2005). Extent of sonochemical degradation and change of toxicity of a pharmaceutical precursor (triphenylphosphine oxide) in water as a function of treatment conditions. Environment International, 31, 207-211.

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Falconer, I. R. (1999). An overview of problems caused by toxic blue-green algae (Cyanobacteria) in drinking and recreational water. Environmental toxicology, 14, 5 12. Fernandez, D., & Ricafrente, M.V. (2010). DOST-PCAMRD supports the PhilHABs program. The PCAMRD Waves, 1(23), 1-6. Fleischer, A., Romero, R., Manning, F., Jeanty, P., & James Jr., A.E. (1991). The principles and practice of ultrasonography in obstetrics and gynecology (4th Ed.). Connecticut, USA: Prentice-Hall International Inc. Food and Agriculture Organization of the United Nations. (2007). Installation and operation of a modular bivalve theory. UK: Author. Goldman, C.R., & Horne, A.J. (1983). Limnology. California: McGraw-Hill Inc. Hallegraeff, G.M., Anderson, D.M., Cembella, A.D., & Enevoldsen, H.O. (1995). Manual on harmful marine microalgae. Place de Fontenoy, Paris: UNESCO. Karlson, B., Cusack, C., & Bresnan, E. (2010). Microscopic and molecular methods for quantitative phytoplankton analysis. Place de Fontenoy, Paris: UNESCO. Jaymalin, M. (1997, May 7). The moribund shellfish industry. The Philippine Star, pp. 1, 17. Jinhui, W. (2005). The ecological engineering of HAB: Prevention, control, and mitigation of harmful algal blooms. Electronic Journal of Biology, 1(2), 27-30. Kinne, O. [Editor], Blaxter, J.H.S., Collier, A.W., Gunkel, W., Helleburst, J.A., & Segal, E. (1970). Marine ecology, v. 1.Environmental Factors, Part 1. London: WileyInterscience. Lee, R.L. (1989). Phycology (2nd Ed.). New York: Cambridge University Press. Lee, S. (2004). Ultrasonic cleaning baths. Retrieved from: http://www.impact test.com/docs/SV050_055HB.pdf Li, H., Huai, X., Cai, J, & Liang, S. (2009). Experimental research on antiscale and scale removal by ultrasonic cavitation. Journal of Thermal Science, 18(1), 65-73. Maclean, J.L. (1977). Observations on Pyrodinium bahamense plate, a toxic dinoflagellate, in Papua New Guinea. Limnology and Oceanography. 22(2), 234-254. Madison Area Technical College, Biotechnology Project. (2005). An overview of sonication. Wisconsin: MATC. Newcombe, G. (2009). International guide manual for the management of toxic cyanobacteria. London: GWRC. Nybakken, J.W. (1982). Marine Biology, an Ecological Approach. New York: Harper & Row. 24

OBrien, W.D. (2007). Ultrasound-biophysics mechanisms. Progress in Biophysics and Molecular Biology. Urbana, IL: Elsevier, 93, 212-255. Olympus (2006).Ultrasonic transducers technical notes. USA: Author. Oyib, D.H. (2009). Control mechanism of algal growth. Everything About Water,11, 40-41. Relox Jr., J., & Bajarias, F. (2003). Harmful algal blooms (HABs) in the Philippines. Retrieved from: http://fol.fs.a.u-tokyo.ac.jp//rtw/TOP/EXabst/019JuanRReloxJr.pdf Ryding, S.O., & Rast, W. (1989). The control of eutrophication of lakes and reservoirs. Cornforth, UK: Parthenon Publishing Group. Sassi, J., Viitasalo, S., Rytkonen, J., & Leppakoski, E. (2005). Experiments with ultraviolet light, ultrasound, and ozone technologies for onboard ballast water treatment. Finland: Julkaisija-Utgivare. Sherman, C.H., & Butler, J.L. (2007). Transducers and arrays for underwater sound. New York: Springer. Song, W., Teshiba, T., Rein, K., Oshea, K. E. (2005). Ultrasonically induced degradation and detoxification of Microcystin-LR (cyanobacterial toxin). Environmental science & technology, 39 (16), 63006305. Using ultrasound to control toxic algal blooms. (2010). Retrieved from http://www.physorg.com/news197715172.html Usup, G., Kulis, D., & Anderson, D. (1994). Growth and toxin production of the toxic dinoflagellate Pyrodinium bahamense var. compressum in laboratory cultures. Natural Toxins, 2, 254-262. Van Iersel, M.M. (2008). Sensible sonochemistry. Eindhoven: Eindhoven University. Villao, R.S. (1988). The red tide menace. Diliman Review, 36(5), 44-45.

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APPENDICES
Appendix A. Summary of Materials and Methods Figure 5.1. Process Flowchart of the Research Study

Procurement of stock culture containing P. bahamense var. compressum (A) Preparation of culture flasks containing P. bahamense var. compressum (n = 24) (B)

Acquisition of an ultrasonic sonicator (D)

Acquisition of Sedgewick-Rafter slide (E) Acquisition of other lab equipment (F)

Labeling, Grouping, and Randomization of Solutions (C) Setting up of Ultrasonic Cleaner (G)

Initial Cell Counting (2 trials) (H)

Exposure of Algae to Ultrasound (Algae vs. Time of Exposure) (I) Final Cell Counting (2 trials) (J) Graphing and Tabulation of Collected Data (K) Statistical Tests (ANOVA and T-Test) (L)

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Appendix B. Formulas, Tables, and Graphs needed for Data and Analysis Table 5.2.1. Table for Analysis of Variance (ANOVA) Source of Variation Treatments Error TOTAL Sum of Squares SSTr SSE SST Degrees of Freedom DFTr DFE DFT Mean Square MSTr MSE Fcalc F tab or Fcrit

n = Total number of samples nk = Number of samples per treatment k = Number of Treatments Tk = Treatment Totals

If Fcalc > Ftab , reject null hypothesis. If Fcalc < Ftab, accept null hypothesis. Table 5.2.2. Table Comparing Initial and Final Cell Density for each Treatment Flask # TRIAL 1 21 TRIAL 2 TRIAL 1 4 TRIAL 2 Table 5.2.3. Table for Correlated T-Test TREATMENT NO: 1 Initial Cell Density Final Cell Density Change In Cell Density (D)

TREATMENT NO: 1 Flask # D D2 TRIAL 1 21 TRIAL 2 TRIAL 1 4 TRIAL 2 SUMMATION ( ) Equations for Correlated T-Test:

27

n = number of sample pairs D = change in cell density = mean difference

= average change in cell density = sum of squares of the difference

If t > tabulated value for t, accept alternative hypothesis, and reject null hypothesis. If t < tabulated value for t, accept null hypothesis, and reject alternative hypothesis. Table 5.2.4. Initial, Final, and Change of Cell Densities of Pyrodinium bahamense var. compressum cultures in Concentration of Algae vs. Time of Exposure Treatment Flask # Initial Trial 1 Trial 2 Change Trial 1 Trial 2 Final Trial 1 Trial 2 Legend: All treatments are exposed with 1 MHz ultrasound C no exposure to ultrasound T-1 1 hr of exposure T-2 1.5 hr of exposure T-3 2 hr of exposure T-4 2.5 hr of exposure T-5 3 hr of exposure T-6 - 3.5 hr of exposure T-7 4 hr of exposure T-8 4.5 hr of exposure T-9 5 hr of exposure T-10 5.5 hr of exposure T-11- 6 hr of exposure C T-1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10 T-11

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Appendix C. Risk Assessment Table 5.3. Risk Assessment Table

Substance/Device/Organism Pyrodinium bahamense var. compressum *NOTE: Medical assistance must be present during experiment of this kind of harmful algae. Risks/Dangerous Effects - Contains saxitoxin (STX), a dangerous toxin. - STX can cause gastrointestinal, respiratory, and neural symptoms. Symptoms will start by vomiting and paralysis. Paralysis will be succeeded by dysphagia, and then death. - Fatalities were usually a result of respiratory failure. There is no specific antidote for PSP, and the manner of treatment is purely symptomatic. Safety Procedures - Before and after handling this algae, hands must be washed with soap and water - Researchers must wear a lab gown, elbow-length puncture-resistant gloves, and boots. - ONLY use mechanical pipetting for algal transfer - No eating, drinking, or applying of cosmetic products during work time. - Excess algae must be disposed in a leak-proof reagent bottle. - All other living things unrelated to the study are prohibited inside the lab that contains the algae. - Precautions: Keep locked up.. Do not ingest. Do not breathe dust. Avoid contact with eyes. Wear suitable protective clothing. If ingested, seek medical advice immediately and show the container or the label. Keep away from incompatibles such as oxidizing agents, acids. - Eye Contact: Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Cold water may be used. Seek medical attention - Skin Contact: Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops. Cold water may be used. - Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention if symptoms appear.

Seawater

- Slightly hazardous in case of skin/eye contact (irritant), ingestion, or inhalation. - May affect behavior (muscle spasicity/contraction, somnolence), sense organs, metabolism, and cardiovascular system. Continued exposure may produce dehydration, internal organ congestion, and coma. Inhalation: Material is irritating to mucous membranes and upper respiratory tract. - When heated to decomposition it emits toxic fumes. - Electrolysis of sodium chloride in presence of nitrogenous compounds to produce chlorine may lead to formation of explosive nitrogen trichloride. Potentially explosive reaction with dichloromaleic anhydride + urea. - Hygroscopic. Reacts with most nonnoble metals such as iron or steel, building materials (such as cement) Sodium chloride is

29

rapidly attacked by bromine trifluoride. Violent reaction with lithium. - Mutagenic for mammalian somatic cells. Lowest Published Lethal Dose (LDL) [Man] - Route: Oral; Dose: 1000 mg/kg

-Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. Loosen tight clothing such as a collar, tie, belt or waistband. Get medical attention if symptoms appear. - Personal Protection: Splash goggles. Lab coat. Dust respirator. Be sure to use an approved/certified respirator or equivalent. Gloves. - Accidental Small Spill: Use appropriate tools to put the spilled solid in a convenient waste disposal container. Finish cleaning by spreading water on the contaminated surface and dispose of according to local and regional authority requirements. - Accidental Large Spill: Use a shovel to put the material into a convenient waste disposal container. Finish cleaning by spreading water on the contaminated surface and allow evacuating through the sanitary system. - Waste Disposal: Waste must be disposed of in accordance with federal, state and local environmental control regulations. - Eye Contact: Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Cold water may be used. Get medical attention. - Skin Contact: Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops. Cold water may be used. - Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious person. Loosen tight clothing such as a collar, tie, belt or

Lugols Iodine

- Hazardous in case of ingestion. Slightly hazardous in case of skin contact ( irritant, permeator), of eye contact (irritant). - Mutagenic for mammalian somatic cells. Classified Reproductive system toxin for females. The substance is toxic to thyroid. The substance may be toxic to blood, kidneys, liver, skin, eyes. Repeated or prolonged exposure to the substance can produce target organs damage. - Potassium iodide (KI) + Fluorine Perchlorate (FClO4) will explode on contact.

30

- Slightly reactive to reactive with oxidizing agents, reducing agents, organic materials, metals, acids. - Acute Potential Health Effects: Skin: Causes skin irritation. It can cause brown stains on the skin. It can be absorbed through the skin. Eyes: Eye contact with liquid causes irritation. Iodine vapors may cause eye irritation. Eye contact with an excessive amount of iodine vapor may also cause blepharitis. Excessive inhalation of iodine vapors may cause respiratory tract, nasal, and mucous membrane irritation. Symptoms may include coughing, tightness in the chest, rhinitis, dyspnea/respiratory distress, coughing, sneezing, pulmonary edema, chemical pneumonitis, edema of the larynx and bronchi, pharyngitis, swelling of the parotid gland, and cachexia. High exposure may lead to lung disease and may also affect behavior/central nervous system (delirium, hallucination, depression, seizure)

waistband. Get medical attention if symptoms appear. - Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical attention. - Hygienic Practices: Avoid contact with eyes, skin and clothing. Wash hands after direct contact. Do not wear product-contaminated clothing for prolonged periods. - Engineering Controls: Provide exhaust ventilation or other engineering controls to keep the airborne concentrations of vapors below their respective threshold limit value. - Personal Protective Equipment: Splash goggles. Lab coat. Gloves. - Spill Procedures: Dilute with water and mop up, or absorb with an inert dry material and place in an appropriate waste disposal container. - Waste Disposal: Dispose of in accordance with all applicable federal, state, and local regulations.

Sonicator

- Safety concerns relating to ultrasound technology are possible noise from the transducer, yet unknown effects upon humans affected by the exposure to ultrasound. -Heat is generated in the transducer if the cooling system fails. - Sonicators are usually constructed of steel, titanium, aluminium or ceramic material. - Sonicators develops noise (if set at high frequencies) that can irritate humans and animals (>1 MHz)

- Do not install the sonicator in a hot/humid area. Place it in an area with proper ventilation - Check the connection cable for damage due to overheating/moisture - If the generator main fuse blows, check the generator for a short circuit, the on/off switch, connection of the supply transformer, printed circuits for blown fuses - Clean out dirt inside and outside of the sonicator. - Wipe/dry ultrasonic cleaner after use.

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Appendix D. Task List Table 5.4. Task List of the Methods in the Research Study ACTIVITY CODE A ACTIVITY DESCRIPTION Procurement of stock culture containing Pyrodinium bahamense var. compressum Preparation of culture flasks containing Pyrodinium bahamense var. compressum Labelling, Grouping, and Randomization of Solutions OBSERVABLE INDICATORS Stock culture obtained from MSI PRECEEDING ACTIVITY B ESTIMATED DURATION 1

Twenty-four 125 mL Erlenmeyer flasks with 100 mL Algal Solutions

24 flasks are labeled acc. to where it belongs Flasks are randomized using CRD Acquisition of an Ultrasonic cleaner ultrasonic sonicator obtained from IMAT Pro Company c/o Mr. Publico Acquisition of an Sedgewick-Rafter Sedgewick-Rafter slide obtained from silde MSI Acquisition of Microscope, pipette, other lab equipment autoclave, stirring (beakers, pipette, rod, regeant bottles, droppers, seawater, Eppendorf microscopes, tubes, Lugols autoclave, etc.) Iodine, etc. Setting Up of Ultrasonic Cleaner is Ultrasonic Cleaner already switched on with 2 flasks in a treatment (Repeated 11 times, one for each treatment) Initial Cell Cell Densities of Counting and each flask are

32

Computation of Initial Cell Density Exposure of Algae to Ultrasound (Algae vs. Time of Exposure or Experiment A) Final Cell Counting

Graphing and Tabulation of Collected Data Statistical Tests

obtained. 2 trials each All 11 treatments are done (All flasks except control are exposed to ultrasound) All 1 mL samples of each flask are counted (2 trials each flask) Graphs and Tables saved in Excel ANOVA, and Correlated T-Test performed with 0.05 level of significance

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Appendix E. Materials Sourcing and Budgeting Table 5.5.1. Table of Materials needed for Experiment and their Costs Quantity Material Needed 1 - Autoclave 15 - Beaker 1 - Microscope 2 Single Channel Pipette 24 125 mL Erlenmeyer Flasks Bolinao Seawater Cheesecloth Cotton Alcohol Lamp CHEMICALS: F/2 medium Lugols Iodine 1 Ultrasonic Cleaner Source Marine Science Institute (MSI) Address Velasquez Street, UP Diliman, Quezon City Contact Esrelita Flores 921-5967 922-3957 Payment Free of Charge

I-MAT Pro Company Marine Science Institute Marine Science Institute

1 - Stock culture of Pyrodinium bahamense var. compressum 4 Sedgewick Rafter Counting Slides

Kentwood Heights, Mariposa Street, Brgy. Crame, Quezon City Velasquez St., UP Diliman, Quezon City

Ramon Publico Esrelita Flores 921-5967 922-3957 Esrelita Flores 921-5967 922-3957

Free of Charge Free of Charge

Velasquez St., UP Diliman, Quezon City

Free of Charge

Table 5.5.2. Table of Transportation and Electricity Consumption Date of Work Consumption Justins Car Round Trip: PSHS to MSI Water Filterer (Uses electricity) Autoclave (Uses Electricity) Commuted: Round Trip: PSHS to MSI Duration or Price 15 minutes 30 minutes 1 hour Taxi Php 80.00 FX to Agham Road (twice) Php 20.00

February 24, 2011

February 25, 2011

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February 28, 2011

March 1, 2011

Microscope Viewing (Uses Electricity) Justins Car Round Trip: PSHS to MSI Microscope Viewing Justins Car

Jeep (UP Ikot) Php 7.00 Jeep (UP to Agham Road) Php 10.00 Total: Php 117.00 2 hours 15 minutes 2 hours PSHS to I-MAT Pro Company 1 hour I-MAT Pro Company to MSI (twice) 30 minutes*2 = 1 hour MSI to I-MAT Pro Company 1 hour MSI to PSHS = 10 mintues Total = 3 hrs. 10 minutes 2 hours 30 minutes 15 minutes 1 hour 12 hours each day

March 4, 2011

Daily

Microscope Viewing Use of Computer (Letter of Documentation Editing) Justins Car Round Trip: PSHS to MSI Ultrasonic Cleaner Lamp (for Algae Control)

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Appendix F. Gantt Charts Figure 5.6. Gantt Chart with Expected Dates of Work
DATE (DAYS) A B C D E F G H I J K L 23-Feb 24-Feb 25-Feb 26-Feb 27-Feb 28-Feb 1-Mar 2-Mar 3-Mar 4-Mar 5-Mar 6-Mar 7-Mar 8-Mar 9-Mar 10-Mar 11-Mar 12-Mar 13-Mar 14-Mar 15-Mar Feb 23 - START, March 15 - END

*For legends, see Appendix D (Task List) Annex E. Network Chart Figure 5.7. Network Chart of the Research Study
A=1d 2 B=1d 3 C=1d 4
D=7d E=1d

1
F=1d

5 6

7
G=1d

8
H=5d

9
I= 5 d

10
J=5d

11
K=1d

12
L=1d

13

36