Overview Fouling is the growth and settlement of marine organisms on submerged surfaces; more than 2500 different marine organisms have been identified to play a role in fouling (Anderson et al. 2003). Moreover, fouling is a major concern to the commercial shipping and recreational boating industries (Champ 2000). Studies have shown that 1 mm increases in the submerged area of the ship (hull) thickness due to fouling increases its drag by 80%, decreasing fuel efficiency by 40% and increasing voyage cost by more than 70% (Champ 2000). Earlier attempts at preventing marine fouling used toxic chemicals, such as arsenic, organo-mercury, DDT, and lead. However, studies in the 1960’sdetermined that when the chemicals were released from applied surfaces, they were harmful and highly persistent in the marine environment (Gough et al., 1994). The results from such studies lead to the voluntary removal of these compounds from the market. Subsequently, alternative chemicals were developed and tested as replacements (Thomas et al., 2000). They found that copped based paint was the most effective against fouling; however,multiple micro algae were strains are copper resistant and can flourish in copper enviornments. Microlage plays a role in subsequent growth of seaweed on hulls (Gough et al., 1994). To overcome such their resistance, organic boosted biocides were added to copper based paints. Irgarol is one of the most commonly used organic boosters in antifouling paints (Thomas et al., 2002). Irgarol, also known as cybutryne, is a protective biocide belonging to the s- triazine group of compounds. Irgarol has a low water solubility (7 mg/L ) and is an effective inhibitor of photosynthesis. Its ability to inhibit photosynthesis makes it an effective, long-life antifouling agent in marine applications. Irgarol, at levels as low as 10 ppb is more effective than other commonly used antifoulants in fresh and saltwater (Readman et al. 1993). Therefore, the use of Irgarol based paints for marine used significantly increased (Ciba-Giegy 1995). Consequently, the widespread use of Irgarol lead to multiple concerns and studies assessing its environmental impact, occurrence, toxicity and fate (Konstantinou and Albanis 2004). The occurrence of Irgarol has been reported in coastal areas worldwide (Readman et al. 1993). The highest ever recorded concentration of Irgarol was 4000 ppt off the coast of Singapore (Basheer et al., 2002). The main source of Irgarol in the environment is leaching from surfaces sprayed or coated with this antifouling agent (Thomas et al., 2002). Comprehensive surveys of coasts in Canada and United States revealed that Irgarol concentration is typically between 1-1816 ppt (Gardinali et al) (Owen et al) (Hall et al). Studies on the uptake of Irgarol into el grass (Zostera marina) and shoal grass (Halodule) was revealed that 9 out of the 10 seagrass samples contained 1-118 ppt of Irgarol (Scarlett et al. 1999). Due to its inhibition of photosynthesis, Irgarol and other herbicides pose a potential threat to coastal environments and microalgae. However, comprehensive understanding of Irgarol’s long-term toxicity and degradation is currently lacking. Irgarol’s persistence in water depends on its photochemical and biological degradation. However, it has been demonstrated that Irgarol does not to partition well into sediments; therefore, it remains in solution (Thomas et al., 2002). According to these studies, Irgarol is considered to be non-biodegradable, and its degradation in seawater and freshwater is slow, with half-life of about 22 and 100 days, respectively. It is believed that environmental factors such as pH and dissolved organic matter can affect the degradation process as they change the chemistry of water (Sakkas et al., 2002). Objective of the Study To the author’s knowledge, this research will be the first to (1) study the effects of photochemical versus biological degradation of Igarol’s 13C and 2H isotope composition using Gas chromatography-combustion/pyrolysis-mass spectrometry (GC-C/P-MS) and, (2) study the effect of varying pH on degradation rate and 13C and 2H isotopic composition on Irgarol. The hypothesis for our study is that (1) the rate of change in 13C and 2H isotopic composition of Irgarol will differ between photochemical and biological degradation, (2) rate of photochemical degradation will exceed biological degradation and (3) pH will affect degradation rate. The result from this study will be able to shine light on the biological and photo- chemical degradation mechanisms and help environmentalists develop new procedures to degrade persistent compounds such as Irgaol faster and efficiently. Progress September 2017- December 2017 The first three months were utilized to analyze previous and current research papers on irgarol. The purpose of this period was to expand our knowledge of irgarol and learn about different experimental procedures used by researches to study the degradation of comparable compounds. We found that research focuses on the metabolites produced by irgarol the degradation process . As per the author’s knowledge, no research was found that specifically focused on the mechanism through which irgarol degraded. Also, little to no information is available on the comparison of biological and photochemical degradation of irgarol. However, we were able some papers that discussed the effects of pH and dissolved organic matter on the degradation of contaminants. The information gathered during this period helped us design our project and its methodology. To facilitate research, it was decided that only one environmental factor (pH) will be tested as it was shown in previous studies to have a greater effect on degradation than dissolved organic matter. We choose to run photo and bio degradation experiments in DI to ensure no organic matter was present. We also decided to test irgarol at a concentration of 1mg/L. in line with prevalent research which use 1 mg/L dissolved in acetonitrile. However, we later changed the concentration to 3.9 uM. As we believed 3.9uM of Irgarol would be enough to be detected by the GC-MS. Since the half-life of irgarol is 22-100 days we decide to run our experiment for 15 days and analyze the sample at 0,1,3,5 and 10 days for each experiment. We chose to use bacteria isolated from the local river (Otanabee river) for the biodegradation experiment since we wanted our experiment to be as eco-friendly as possible and prevent the addition of new bacteria in a local water source that can harm to environment and its species. We choose to use a sun simulator to speed up the photo degradation experiment as simulation will provide a consistent amount of sunlight. In addition, we choose to use plastic cups covered with soran wrap to simulate a lake/ river condition. For the biodegradation experiment, we choose to use opaque sterile cups to ensure no contamination would be present. In order to prevent radiation contamination, we decided to cover them with aluminum foil. We began training on the GC/MS with caffeine as a substitute for our contaminant. We ran caffeine solutions through GC-MS couple with combustion and pyrolysis column to become familiarized with the GC-MS and its software. We succeed with our test as we were able to get a caffeine peak in the FID detector and a 13C and 2H isotopic composition of caffeine. January 2018 We prepped a 3.9 uM solution of irgarol in methanol from a 100 ppm stock solution. We ran it through the GC-MS combustion column using the conditions used in previous studies. The GC-MS failed to detect the irgarol. Therefore, we ran a higher concentration of irgarol (0.013 mM) in acetonitrile yet failed to detect a peak the GC-MS. Lastly, 100 ppm solution of irgarol in methanol was tested in the GC-MS combustion column and no result were achieved. To crack down the problem, we ran 0.16 uM and 0.33 uM of 4-octylphenol solution through the GC-MS, but no peaks were detected. We believed that the combustion column needed to be re- ionized. Therefore, we ran the re- ionized procedure recommended by the manufacturer. Next day, we ran a 0.026 uM irgarol in methanol solution through the GC-MS, but still could not detect a peak. We believe that both irgarol and 4-octylphenol must have degraded as the stock solutions used were old. We tried changing the parameter of the GC-MS to run for longer period and higher temperature making sure enough time was given for compounds to pass through the column. We also tried dissolving Irgarol in different media, but no peaks were identified. Lastly, we decided to make new stock solutions and try again. Need to do In order to complete the research, we need to make a new stock solution. The new solution will be tested using the GC-MS, and if peaks are detected, then a 100 ml of irgaol stock solution will be made. The stock solution will be used to make two irgarol solutions at pH 5 and 8 insodium acetate. The water will be filtered through a 0.22 um pore filter in an amber glass bottle. The filter will be then placed in a glass bottle containing ultra-pure water in the dark for 12 hours to allow acclimation of bacterial communities at room temperature (21°C). The isolated bacteria will then be placed in media containing nutrition broth in sterilized glass bottles that later will be used to spike the biodegradation experiment solution. To ensure no radiation and no contaminates are presents, sterilized plastic cups covered will aluminum foils will be used as test containers for the biodegradation experiment. The prepared pH 5 and pH 8 solutions will be placed in two cups and will be spiked with the bacteria isolated from the Otonabee river. The cups will be wrapped with aluminum foil and will kept at room temperature for 15 days. On the other hand, for the photo-degradation experiment, two plastic cups with the pH 5 and pH 8 irgarol solutions will be covered with saran wrap to simulate lake conditions. The cups will be placed in a sun simulation for 15 days. The solution will receive constant amount of radiation. The test samples from the photo- degradation and biological degradation experiments will be analyzed by gas chromatography-combustion/pyrolysis mass spectrometry for 13C and 2H isotopes. The column used will be HP5ms coated capillary column with 0.25 um film thickness, 0.32 mm i.d. x 30 m length installed to the injector with 250 °C in the splitless mode with a constant helium carrier flow at 1.3 mL/min.2 Depending upon the result from the test solution, the run time and temperature may be changed in order to get better peaks.