Ens 203 Final Paper

An Assessment of the Effects of Solar UV-B Radiation on Turbinaria conoides and Padina minor

by Renz L. Salumbre MS Zoology

October, 2008

TABLE OF CONTENTS Table of Contents......................................................................................................... i List of Figures............................................................................................................... iii I. Introduction................................................................................................................ 1 A. Background of the Study........................................................................................ 1 B. Statement of the Problem...................................................................................... 2 C. Objectives of the Study.......................................................................................... 3 D. Significance of the Study.......................................................................................

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E. Scope and Limitation.............................................................................................

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II. Review of Related Literature.................................................................................... 5 A. The Ozone Layer.................................................................................................... 5 B. Global Warming.....................................................................................................

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B.1 The Process of Global Warming........................................................................ 5 B.2 Biological Impacts of Global Warming...............................................................

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C. Solar Ultraviolet Radiation..................................................................................... 7 C.1 Classification of Ultraviolet Radiation Wavelengths........................................... 7 C.2 Factors that Affects Ultraviolet Radiation........................................................... 8 C.3 Measurement and Evaluation of Solar Radiation..............................................

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C.4 Effects of Ultraviolet Radiation........................................................................... 9 III. Methodology............................................................................................................ 11 A. Study Area.............................................................................................................. 11 B. Algal Species.........................................................................................................

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C. Collection of Data..................................................................................................

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C.1 Abiotic Factors................................................................................................... 12 C.2 Collection of Algal Specimen............................................................................. 13 D. Data Analysis......................................................................................................... 13 i

D.1 Analysis of Gross Morphology...........................................................................

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D.2 Analysis of Ultrastructure................................................................................... 13 D.3 Comparison with Control Samples....................................................................

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D.4 Computations..................................................................................................... 14 D.5 Statistical Analysis............................................................................................. 14 IV. References.............................................................................................................. 15 V. Appendices............................................................................................................... 17

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List of Figures Figure 1. The Process of Solar Radiation Entering the Earth............................

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Figure 2. Different Types of Atmospheric Intervention when Solar Radiation reaches the Earth’s Atmosphere........................................................................

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Figure 3. Radiation Spectrum of Ultraviolet Rays..............................................

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Figure 4. Selected Sites of Study.......................................................................

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Figure 5. Preserved Specimens of Turbinaria conoides (A) and Padina minor 20 (B)....................................................................................................................... Figure 6. The Azide-Winkler Method for Measuring Dissolved Oxygen 21 Content...............................................................................................................

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Chapter I INTRODUCTION A. Background of the Study Stratospheric ozone depletion is of great concern as of the moment. Recent findings demonstrate that the ozone layer established in the stratosphere retains approximately 95% to 99% of the sun’s ultraviolet radiation from striking the earth (Sparling, 2002). The sun produces both visible and invisible rays. Invisible rays known as ultraviolet-B (UVB), which is 290-320 nanometers, cause most of the alleged environmental problems such as the greenhouse effect and global warming. Affirming this, Kraffert (1998) pointed out that UVB is more hazardous than any other type of ultraviolet radiation. Biologically harmful UV-B reaches the earth’s surface in amounts inversely proportional to the concentration of atmospheric ozone. Some factors that impede UV-B release are urban air pollution, suspended particulates, aerosols, and ozone in the troposphere, as well as by stratospheric ozone. UV-B has harmful effects on a wide range of biological systems. It can cause DNA damage which is proportional to the intensity and duration of exposure; unfortunately, small, delicate organisms suffer more severe damage than large robust species (such as humans and other mammals). UV-B, moreover, impairs the growth and photosynthesis of certain plants and impairs the motility and reproductive capacity of phytoplankton. Change is evident in the composition of phytoplankton in aquatic ecosystems, some of which are already under UV-B stress. Further increases in UV radiation are expected to cause detrimental effects including disruption of specific food chains in these ecosystems. In addition, marine phytoplankton metabolizes a great deal of atmospheric CO2; reduction of phytoplankton will decrease the uptake of CO2 and thus aggravate the greenhouse effect. In

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short, the ecological effects of increased surface level UV-B radiation, although not fully predictable, are likely to be widespread and harmful (Hader & Figueroa, 1997). Macroalgae and sea grasses are important biomass producers in aquatic ecosystems. In contrast to phytoplankton, most of these organisms are sessile and cannot avoid exposure to solar radiation at their growth site. Recent investigations showed a pronounced sensitivity to solar UV-B radiation with the effects being demonstrated throughout the top 10 to 15 meters of the water column. In addition, photosynthesis in the symbiotic algae is impaired, resulting in reduced organic carbon supply. The succession of algal communities is controlled by a complex array of external conditions, stress factors and interspecies influence (Hader & Figueroa, 1997).

B. Statement of the Problem The increasing awareness regarding the environmental impact of global warming has initiated a considerable amount of response by various specialist whether it be economics, chemistry, physics, or ecology. This said awareness has given rise to the need of more studies that shall provide a modicum of unmitigated evidence in regard to the destruction of biological systems. As the ozone layer gets depleted due to various factors, most studies have focused on the impact of the regressing quality of the ozone layer and how it affects various species. As such, this paper will take into account the alleged effects of solar UV-B radiation on algal species in an in vivo setting as opposed to the one originally proposed by Holzinger & Lütz (1993) whose experiment with ultraviolet irradiation on algae was achieved in a laboratory setting. Algae present in both Pangasinan and Batangas during the summer season will be collected while a control group is established and will be maintained.

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Afterwards, the said group of algae will be subjected to a thorough examination of the gross morphology and, subsequently, the ultrastructure using scanning electron microscopy.

C. Objectives of the Study In this particular study, two representative species of algae both present in Pangasinan and Batangas will be examined. Simply put, the purpose of this study is to give evidence that the regressing quality of the ozone layer has a considerable effect on algal communities. The basis of the study shall consider the visible changes that may have result due to significant levels of UV-B radiation. The main objective, therefore, is to formally recognize, if any, the effects of solar UV-B radiation on algal communities of Padina minor and Turbinaria conoides in Bolinao, Pangasinan and Calatagan, Batangas. The specific objectives are the following: first, to identify the degree of destruction brought about by high levels of solar UV-B radiation on the aforementioned species of algae in terms of their gross morphology and ultrastructure; second, to compare the gross morphology and ultrastructure of the above species of algae found in both sites; third, to corroborate the claim that algae are greatly affected by increasing levels of UV-B radiation; lastly, to correlate the results on global warming and the destruction of biological systems.

D. Significance of the Study Once this study is accomplished, it is expected that it will add to the growing repository of knowledge regarding the impact of global warming. It is expected also to help people to be aware of the increasing destruction brought about by anthropogenic factors which in turn cause global warming. This study will also corroborate the fact that global warming is altering 3

ecosystems. As the ozone layer is depleted by various factors, plant life or plant structure is greatly altered in reference to their habitats. This study is also expected to emphasize the biologically-harmful effects of UV-B radiation. It is also significant to compare the effect of UV-B radiation on the study area (which includes the Northern and Southern part of Luzon) as well as its effects on the representative algae so as to serve as a future reference for doing similar studies.

E. Scope and Limitation This study will cover, in general, the harmful effects of UV-B radiation on biological systems particularly on algal communities. UV-B radiation, as mentioned above, has been observed to be detrimental to plant life compared to other members of the spectrum. The algal materials that will be scrutinized are Padina minor and Turbinaria conoides due to the seasonality of algae and the availability of these algae at Bolinao, Pangasinan and Calatagan, Batangas. The responses of algae to UV-B radiation will be evaluated by observing the gross morphology and the ultrastructure of these algae with special reference to chloroplast and mitochondria and the concomitant occurrence or destruction of structures that are likely related to UV-B stress. The abiotic parameters that will be taken into account are the following: pH levels, salinity, temperature, water quality and dissolved oxygen content. Field work shall be accomplished during the summer season while experimentation shall be carried out through the remainder of the year.

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Chapter II REVIEW OF RELATED LITERATURE

A. The Ozone Layer The stratospheric ozone layer (Figure 1) is practically a shield that protects the earth from the harmful rays of the sun. The ozone layer, before, is actually thick enough to filter off the harmful UV-B rays. Solar ultraviolet light splits the ozone’s O3 molecules into O2 and atomic oxygen, which have a very high potential for recombination, which, in turn, reinforces the ozone layer; this ozone-making process protects the earth from the cell-damaging effects of UV-B radiation (UCUSA, 2005). In reality, the biologically harmful ultraviolet

radiation

levels reach the earth in very fractional levels. Unfortunately, due to the depleting quality of the ozone layer and the abundance of atmospheric pollutants, such as fluorocarbons and nitrogen oxide, the ozone layer cannot give the protection it was made for and has allowed the transmission of injurious ultraviolet radiation (Acra, Jurdi, Mu’allem, Karahagopian & Raffoul, 1990). B. Global Warming B.1 The Process of Global Warming Global warming has been pointed out to be the major consequence of the regression of quality of the ozone layer. Global warming happens when greenhouse gases are trapped into the earth, specifically, in the lower atmosphere. It is called as such because when the sun’s rays enter the earth’s atmosphere, the heat from these rays cannot escape. Global warming has been considered to be the driving force behind drastic climate changes (ESS, 2000). The pprocess of global warming is as follows:

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The sun’s rays reach the earth in the form of light waves. This results to the earth heating up. The energy that came from the sun is involved in the said heating-up process and some are re-radiated back into space in the form of infrared waves. It is normal for some of these infrared waves to be trapped into the earth because it keeps the earth’s temperature constant enough to be feasible for living. Some problems have arisen in connection with the increasing number of pollutants that is making the atmosphere thin enough. Usually, these pollutants come from industrial sources. Moreover, human-caused carbon dioxide and other greenhouse gases attack the ozone in bulk resulting in the entrapment of large amounts of infrared waves that should have been re-radiated back into space. The results to the earth’s increasing temperature (Gore, 2006). B.2 Biological Impacts of Global Warming Without doubt, global warming has a lasting impact on all life forms. The world is experiencing a wave of mass extinction of various animals around the world such that the rate of extinction is a thousand times higher than the normal background rate. The factors that contribute to this devastating effect are the same factors that contribute to the phenomenon of climate change; an example of the correlation between these two events is the destruction of the Amazon rain forest which has resulted in species being driven to their extinction and the addition of carbon dioxide to the atmosphere. Other effects of global warming are sea level ris, drastic weather changes and marine life destruction (Gore, 2006). It is a now universally-accepted notion that global warming is causing coral bleaching. In a 1998 study, Buccheim provided evidence that ultraviolet radiation is the leading factor causing coral bleaching and coral mortality. In the study, coral bleaching occurred during periods of high heat and high penetration of short wavelength ultraviolet radiation. In connection with this occurrence, Gore (2006) mentioned that during 2005, considered to be 6

the hottest year, a massive loss of coral reefs occurred. While earlier, in 1996, the second hottest year, 16% of the world’s coral reef was lost. Both mention and affirmed the fact that global warming through increased ocean temperatures mattered greatly in the mortality of corals. Not only are corals affected but other marine life forms are affected too. The threat of marine destruction arises from the uncontrolled growth of carbon dioxide; one-third of whcih end up sinking into the ocean, thus increasing the acidity of water. This “chemical change” of the world’s ocean has given rise to dead zones characteristically devoid of marine life. Others have caused algae to bloom in waters which are already polluted. These algae can grow so big so as to inhibit not only the natural process but also business and other man-made operations.

C. Solar Ultraviolet Radiation C.1 Classification of Ultraviolet Radiation Wavelengths According to Acra et al (1990), the electromagnetic radiation emitted by the sun’s rays can be subdivided into many wavelengths. The said classification is based on the capability of ionizing atoms in radiation-absorbing matter. These are: a) ionizing radiation such as X-rays and gamma rays; and b) non-ionizing radiation such as ultraviolet radiation, visible light and infrared radiation. Solar ultraviolet radiation in the electromagnetic spectrum ranges between 100 nanometers to 400 nanometers. Ultraviolet rays are shorter than visible light but longer than soft X-rays, which are still subdivided according to their biological effects with UV-B having the most dangerous and harmful effects (Figure 3).

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C.2 Factors that Affect Ultraviolet Radiation Diffey (1990) gave the following as factors that affects terrestrial ultraviolet radiation (Figure 2) : a) time of day - about 20% to 30% of total ultraviolet radiation is experienced during midday of summer with approximately 75% occurring between 0900 hours to 1500 hours (9 am to 3 pm); b) season - ultraviolet radiation is seasonally dependent on temperate regions; however, seasonal variation is less when nearer the equator; c) geographical latitude - ultraviolet radiation decreases with increasing distance from the equator; d) clouds - clouds reduce solar radiation levels, although, the changes in the ultraviolet spectrum are negligible; lastly, f) altitude - an increase in altitude increases the levels of ultraviolet radiation; whereas if an area has a low altitude, e.g. sea level, there is a low level of ultraviolet radiation. C.3 Measurement and Evaluation of Solar Radiation Stickler (2007), in his educational brief for the National Aeronautics and Space Administration, enumerated the following equations for measuring the level of solar radiation. The Inverse Square Law is a routine measure of the decrease of radiation intensity due to an increase in distance from the source of radiation. The formula is (Formula 1): I = E (4∏R2) (4∏r2) where I is the Irradiance at the Surface of the Outer Sphere; E is the Irradiance at the Surface of the Object, which, in this case is the sun; 4∏R2 is the surface area of the object; and 4∏r2 is the surface area of the outer sphere. The irradiance of an object can be obtained using the Stefan-Boltzman Law. However, this Law is applicable only if the temperature of the object is known. The formula is (Formula 2): E = IδT4

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where I is the Emissivity of the Object; δ is the Stefan-Boltzman constant which is equivalent to 5.67x10-18 W/m2K4; whereas T is the Temperature of the Object. The emissivity of an object is the factor of how well a surface can absorb and emit energy. More precisely, it is the ration of the emitted power of the body to that of a blackbody at the same temperature. Emissivity ranges from 0 to 1: when emissivity is equal to 1, then the object is said to be a perfect radiator and absorber such as a blackbody. When the emissivity is equal to 0, the object is said to be a perfect reflector (Giambattista, 2007). Another equation which shall be of use concerns Insolation (Formula 3). Its formula is: I = S cos Z where I is the Insolation which is the rate at which direct solar radiation is incident upon a unit horizontal surface at any point on or above the surface of the earth; S which is approximately equivalent to 1000 W/m2 (this value is not absolute since it is the solar insolation during clear days only); and Z is the Zenith Angle which is the angle from the zenith to the sun’s position. The Zenith Angle (Formula 4) can be computed through the following equation: Z = cos-1 (sinΦ sinσ + cosΦ cosσ cosH) where Φ is equal to the latitude; σ is the solar declination angle; and H is the hour angle which can be computed by the following equation (Formula 5): H = [15º x (Time-12)] C.4 Effects of Ultraviolet Radiation Sampson and Cane (1999) have concluded that intensifying levels of UV-B greatly affects flower production: There is a considerable amount of delay of flowering, and flower production is greatly diminished in some plants which they have tested. However, they have observed that there was no overall effect on pollen and nectar production.

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In Newton, Tyler and Slodki (1979), blue-green algae (cyanobacteria) was subjected to UV-B stress. It was observed that cyanobacteria’s nitrogen-fixing enzyme system seem to be highly sensitive to UV-B damage; and that inhibition of nitrogenase activity can take place even other physiological functions are suppressed. Consequently, the have concluded that the measure of acetylene reduction activity in nitrogen-fixing systems may provided a simple biochemical assay for evaluating the biological effects of UV-B. Mehta and Hawxby (1977) used UV radiation in a laboratory-based experimental setup. They used various intensities of UV radiation to achieved bacteria-free algal culture. They concluded the following: there was not bacterial growth on algal cultures inoculated on nutrient agar for 64 hours; algal pigments were not affected by UV radiation; phycocyanin was not inhibited; and that growth requirements were not at all altered. Overall, they concluded that UV radiation is quite effective in obtaining and maintaining axenic algal cultures. Morton and Derse (1968), however, observed that use of gamma radiation (as opposed to ultraviolet radiation) on algae was effective in controlling growth where algae are unwanted. They also recommended that a dosage of 100 krads to 150 krads is needed in a laboratory set-up. Grad (2003) recommended that when studying aquatic organisms which are treated with high levels of radiation, the experimentation should be subjected to frequent monitoring. She claimed the above because recent studies have concluded that ultraviolet tolerance, once assessed shortly after exposure, may be overestimating the true tolerance of the organism.

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Chapter III METHODOLOGY

A. Study Area Field works will be conducted by the start of the summer season starting from March ending up to July of 2008. The sites of study are beaches found in Calatagan, Batangas and Bolinao, Pangasinan (Figure 4). Bolinao (16º 23’ 0.83” North; 119º 53’ 87” East) is situated at the northwestern tip of Pangasinan and the Lingayen Gulf. It is bounded on the north and west by the China Sea, on the south by the rolling hills and plateaus of Bani, and on the east by the Kakiputan Channel. Calatagan (13º 49’ 53.34” North; 120º 38’ 8.04” East) in Batangas comprises teh Calatagan Peninsula between the South China Sean and Balayan Bay.

B. Algal Species Turbinaria conoides and Padina minor are the only algae that shall be of importance to this study because these algae are present at both sites at the same months. Turbinaria conoides (Figure 5A) have an erect thalli, yellowish brown to dark brown in color. It thrives mostly on sandy-corally bottom with preference to reef portions not exposed to water turbulence (Trono, 1997). Padina minor (Figure 5B)is a blade flabellate, yellowish brown to whitish color, up to seven centimeters high. They are attached to solid substrates on reef flat and upper subtidal zone. They grown on the inner reef flats and on tidal pools on the outer portions of reef flats (Trono, 1997).

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C. Collection of Data C.1 Abiotic Factors This study shall observed the following abiotic factors as its parameters: pH, temperature, salinity and dissolved oxygen content, water depth and level of solar UV-B radiation. To measure the temperature, a room thermometer in centigrade will be used. The thermometer will be immersed in sea water and the temperature will be recorded. The process will be repeated thrice at a 30-minute interval. The average of the recorded temperature on a single field work day will be the considered as the temperature of that particular day. The pH will be measured by employing a pH meter. The device will be immersed in sea water and the numerical value will be recorded. The process will be repeated thrice at a 60minute interval. A salinity meter will be used to record the level of salinity in sea water. The salinity meter will be immersed in sea water and the numerical data recorded. This process will be repeated thrice at a 60-minute interval. For the dissolved oxygen content, the Azide-Winkler method will used to gather data (Figure 6). Water depth will be measured using a meter stick or yard stick (or similar equipment) Measurement of water depth will be repeated three times and the average of the data recorded. As for the level of solar UV-B radiation, a radiometer shall be used to record the amount of radiation for a particular study site. This will be repeated hourly and fluctuations in

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the level of UV-B radiation recorded. The radiometer to be used should be specialized for field works. Aside from the above, the weather shall also be observed. C.2 Collection of Algal Specimens Field works shall be conducted from 9:00 am to 3:00 pm for 3 days on consecutive weekly trips that will take place starting in March and ending in July. Parameters mentioned above shall be employed. A number of algal specimens shall be collected with the recommended minimum of 10 samples per specimen per collection day. Collected specimens shall be placed in containers or in plastic bags (zip locks). For larger samples, these will be placed inside containers with salt water or formalin or Bouin’s solution. Small samples shall be placed in zip locks. However, before they are placed, a photograph must be taken before preserving the samples.

D. Analysis of Data D.1 Analysis of Gross Morphology Gross morphology of the algal specimens shall be observed in either the field of study or at a laboratory. Destruction of leaf and other part of the thallus will be observed. Its length and width will be measured using a foot rule. The surface will be categorized as rough, smooth; shiny or slightly opaque. The intensity of coloration shall also be taken into consideration. D.2 Analysis of Ultrastructure Employing scanning electron microscopy, the degree of destruction on the chloroplast and mitochondria will be observed on the two algal specimens at two different sites. Photomicrographs will be ordered afterwards it shall be recorded and analyzed. 13

D.3 Comparison with Control Samples In order to establish a reliable analysis of the effects of solar UV-B radiation, a control group will be established consisting of Turbinaria conoides and Padina minor cultivated in vitro. These two laboratory samples shall then be subjected under ultraviolet bulbs. Gross morphology and ultrastructure of the two mentioned algae shall be analyzed and then compared to those found in the study sites. D.4 Computations The amount of radiation shall be observed using a solar radiometer. However, in order to justify the accuracy of the numerical data produced by the solar radiometer, the Inverse Square Law equation may be employed (Formula 1). D.5 Statistical Analysis Using a statistical software package, the data gathered will be analyzed using T-test for the comparison of the actual difference between the two means in relation to the variation in the data. Also, One-Way Analysis of Variance shall estimate the variability between groups compared with variability between groups (Mendenhall, Beaver & Beaver, 2006).

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Chapter IV REFERENCES Acra, A., N. Jurdi, A. Mu’allem, Y. Karahagopian & Z. Raffoul. Water disinfection by solar radiation : Assessment and application. Retrieved at http://almashriq.hiof.no/lebanon/ 600/610/614/solar-water/idrc. Bischof, K., G. Peralta, G. Kräbs, W.H. van de Poll, J.L. Péres-Lloréns & A.M. Breeman. 2002. Effect of solar UV-B radiation on canopy structure of Ulva communities from southern Spain. Journal of Experimental Botany Vol. 53, No. 379, pp. 2411-2421. Buccheim, J. 1998. Coral reef bleaching. Retrieved at http://www.marinebiology.org/ coralbleaching.htm. Department of Ecology. 2006. How to measure dissolved oxygen. Washington State : USA. Retrieved at http://www.ecy.wa.gov/programs/wq/plants/management/joysmanual/ 4oxygen.html. Diffey, B.L. 1990. Solar ultraviolet radiation effects on biological systems. Retrieved at http://www.ciesin.columbia.edu/docs/001-503/001-503.html. Essential Systems and Services for EH&S and Crisis Management. Global warming. Retrieved at http://www.ess-home.com/news/global-warming/ozonedepletion.asp#thumb. Giambattista, A., B.M. Richardson & R.C. Richardson. 2007. College physics (2nd ed.). McGraw-Hill : USA. Gore, A. 2006. An inconvenient truth : The planetary emergence of global warming and what we can do about it. Rodale : New York, USA. Grad, G. 2003. UV damage and photoreactivation : Timing and age are everything. Retrieved at http://www.findarticles.com. Hader, D.P. & F.L. Figueroa. 1997. Photoecophysiology of marine macroalgae. Photochem. Photobiol. No. 66 Vol. 1-14. Holzinger, A. & C. Lütz. 1993. Algae and UV irradiation : Effects on ultrastructure and related metabolic functions. Retrieved at http://cat.inist.fr/? aModele=afficheN&cpsidt=17547275. Mehta, R. & K. Hawxby. 1977. Use of ultraviolet radiation to achieve bacteria-free algal culture. Proc. Okla. Acad. Sci. Vol. 57 pp. 54-60. Mendenhall, W., R.J. Beaver & B.M. Beaver. 2006. Introduction to probability and statistics. Thomson Brooks/Cole : Singapore. 15

Morton, S.D. & P.H. Derse. 1968. Use of gamma radiation to control algae. Wisconsin Alumni Research Foundation. Vol. 2, No. 11 p. 1041. Newton, J.W., D.D. Tyler & M.E. Slodki. 1979. Effect of Ultraviolet-B (280 to 320 nm) radiation on blue-green algae (cyanobacteria), possible biological indicators of stratospheric ozone depletion. Applied and Environmental Microbiology. Vol. 73. No. 6, pp. 1137-1141. Sparling. B. 2002. Stratospheric ozone depletion. Morgan Hill : California. Stickler, G. Solar radiation and the earth system : Relating solar radiation physics to earth & space science concept. National Aeronautics and Space Administration. Retrieved at http://education.gsfc.nasa.gov/experimental/all98invProject.Site/Pages/ science-briefs/ed-stickler/ed-irradiance.html. Trono, G.C. 1997. Field guide & atlas of the seaweed resources of the Philippines. Bookmark : Makati City, Philippines. UCSUSA.org. 2005. The science of stratospheric ozone depletion. Union of Concerned Scientists. Retrieved at http://www.ucsusa.org/global_warming/science/the-science-ofozone-depletion.html. Other Internet Sources http://asd-www.larc.nasa.gov http://landsat.usgs.gov http://msp.rmit.edu.au/Article_01/02.html http://www.philippinetraveler.com http://www.com.univ-mrs.fr/IRD/atollpol/ecorecat/images/halimac.jpg http://www.usep.edu.ph http://www.ecy.wa.gov http://www.wikipedia.org

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APPENDICES Appendix A

Fig. 1. The Process of Solar Radiation Entering the Earth

Figure 2. Different Types of Atmospheric Intervention Occurring when Rays of the Sun reaches the Earth’s Atmosphere 17

Figure 3. Radiation Spectrum of Ultraviolet Rays

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Appendix B

Figure 4. Selected Sites of Study in Norther and Southern Luzon

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Appendix C

A

B

Figure 5. A) Turbinaria conoides preserved and B) Padina minor preserved

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Appendix D

Figure 6. The Azide-Winkler Method for Measurement of Dissolved Oxygen Content

The Azide-Winkler Method is done through the following procedure (Washington State Department of Ecology, 2006).

1. A glass stoppered 300 ml bottle was filled with sample water. 2. 2 ml of manganese sulfate was immediately added to the collection bottle by inserting the calibrated pipette just below the surface of the liquid and squeezing slowly avoiding bubble 21

formation. Oxygen is introduced into the sample if the reagent is added above the sample surface. 3. 2 ml of alkali-iodide-azide reagent was added in the same manner. 4. The bottle was stoppered and was made sure no bubbles were formed. The sample was mixed by inverting the bottle several times. Again, the bottle was checked if air bubbles were formed. If oxygen is present, a brown to orange cloud of precipitate will appear. This is called the floc. When the floc has settled to the bottom, the sample was again mixed by inverting the bottle several times. Again, the precipitates were allowed to settle again. 5. 2 ml of concentrated sulfuric acid was added by using a pipette held just above the surface of the sample. The bottle was again stoppered and inverted several times in order to dissolve the floc. At this point, the sample can be considered “fixed” and ready for storage. Sample can be stored up to 8 hours if kept in a cool dark place. As an added precaution, distilled water was squirted along the stopper with the cap bottle wrapped with aluminum foil and secured by a rubber band.

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