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stockholm junior water prize entry 2010 novel biodegradation of expanded polystyrene and reduction of toxicity in polystyrene-contaminated environment via microbial adaptation alexandre allard danny luong québec canada

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i abstract excess of expanded polystyrene eps is a rising environmental problem that threatens not only marine life but also the general quality of water recent studies have shown that eps is capable of leaching toxic molecules such as styrene or bisphenol a into water consequently a novel biotechnological method was developed to biodegrade eps in a liquid environment three strains of microorganisms streptomyces griseus pseudomonas putida and pseudomonas fluorescens were isolated and subjected to an enrichment procedure to select for an adaptation to eps the three strains were able to synergically and optimally biodegrade 69.5 of the mass of the eps in 2 weeks by producing enzymes and biosurfactants this novel method could be applied in water bioremediation or industrial enzymatic degradation to reduce the amount of eps in rivers and oceans and to increase water s drinkability ii table of contents i abstract iii keywords 1 2 2 2 2 3-4 4-8 8-11 12-13 13-14 14-15 15 ii table of contents iv abbreviations and acronyms v acknowledgements 1 introduction 3 results 4 discussion 6 references 7 annexes 2 materials and methods 5 conclusions 1

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iii keywords expanded polystyrene synergy biodegradation bioremediation iv abbreviations and acronyms lb luria-bertani broth eps expanded polystyrene bpa bisphenol a ppm parts per million exp experimentals ctl controls v acknowledgements we would like to thank our professors daniel guillemette martin dupré and anna dera for opening the chemistry and microbiology labs in our school and for providing to us the materials needed for the experiment special thanks to alexandre lemieux réda bensaidane antoine dussault and annie-jade samson for their support during the entire project vi biography my name is alexandre allard and this year i will be attending mcgill university in the pre-medicine program my hobbies include piano and sound mixing i volunteer in my community and at my cégep i was a member of the student council as public relations officer i am often on stage as master of ceremony at my school shows or as a member of the improvisation team this year my project was awarded a silver medal at the canada wide science fair as well as participation to the milset science fair in slovakia next year later on in life i intend to specialize in microbiology and immunology and research new ways of curing and preventing human diseases my name is danny luong and i am currently in cegep grade 12 equivalent my strong interests in microbiology and in environmental sciences made me decide to study in environmental bioengineering next year i hope to someday contribute to scientific research by developing new and greener methods to preserve environments waters soils etc this year my project was awarded several prizes such as best-in-fair at the regionals bronze medal at the provincials and a silver medal at the nationals i will also be part of canada s delegation to milset 2 esi 2011 in slovakia next year.

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1 introduction polystyrene is a very common type of plastic worldwide annual production was approximately 16 million metric tons in 2008 [1 which is a polymer consisting of long chains of styrene c8h8 monomers formed by polymerization [2 one of its most common forms is expanded polystyrene eps generally called styrofoam® which is produced by injecting pentane c5h12 as a blowing agent during the polymerization phase [2 eps worldwide annual production of approximately 7 million metric tons in 2008 [1 is mainly used in packaging or insulation applications in the pharmaceutical food and construction industries [1 2 currently used eps is rarely recycled because it is either contaminated with food grease or chemicals which makes the recycling process not economically viable [3 also since eps takes a lot of space transportation to the nearest facilities is not efficient and contributes to global warming [3 according to the us environmental protection agency the recycling rate of eps in the united states is less than 1 [3 consequently used eps is either stored in landfills on shores or dumped in oceans [3 5 6 for example in japan only 150 000 tons of eps is annually stored on the shores of the pacific ocean [4 5 threat to marine environment most of the eps on the shores or in rivers will eventually end up in the eastern garbage patch or the trash vortex in the pacific ocean by getting swept by currents from the north pacific gyre figure 1 [6 eps in oceans will naturally break down into smaller pieces [5 that could potentially be ingested by at least 267 species in the marine environment especially fish and sea turtles thus obstructing their digestive tracts or killing them [7 recent studies in japan from nihon university reported at the 238th national meeting of the american chemical society that eps is capable of decomposing and releasing toxic molecules such as styrene and bisphenol a bpa into the pacific ocean in less than one year [4 5 consequently eps in water is not as chemically stable as many once thought past research has demonstrated that bpa an 3

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endocrine disruptor [8 could potentially harm mollusks amphibians fish and crustaceans even at very low concentrations in water by affecting their reproductive systems [8 also the international agency for research on cancer iarc classified styrene as a potential human and animal carcinogen [9 thus eps and these two chemical compounds are considered as water pollutants negatively affect its drinkability and are harmful to marine environments [8 10 microbial adaptation hypothesis and purpose scientific literature has shown that microorganisms especially bacteria are capable of adapting through stress to various chemical compounds such as pesticides [11 antibiotics [12 and pollutants like ethanol or toluene [13 in this experiment it was hypothesized that microorganisms could adapt to a highly concentrated eps environment and use the polymer as a carbon source a novel biotechnological approach is proposed in this paper as an alternative to industrial and experimental degradation methods gasification pyrolosis and recycling with d-limonene that are either expensive or not environmentally friendly because of the use of extreme heat or organic solvents [14 these traditional methods are based on chemical approaches the purpose of this experiment was to develop an optimal method to degrade eps efficiently ecologically and inexpensively for industrialized and third-world countries by isolating strains of microorganisms and adapting them to the polymer with an enrichment procedure in scientific literature only one article was found on eps biodegradation but was unsuccessful in degrading eps with various fungi [15 2 materials and methods n.b all materials used were sterilized and all manipulations save for the soil sampling were carried out in an aseptic environment the eps in this experiment was purchased from home-depot and was disinfected with 70 isopropanol a comparison of the weight of the eps before and after disinfection demonstrated that the isopropanol did not degrade or affect the eps quantification of polystyrene degradation and controls degradation was measured by comparing the initial weight of the test tubes pyrex® vistatm 200mm containing the microbial cultures luria-bertani medium lb per l tryptone 10g yeast extract 5g nacl 10 g ph 7.0 and eps to the weight of the test tubes after incubation tris buffer was used for ph stabilization 50 mm the weight was measured using analytical balances sartorius 4

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la120s with an error of ± 0.0002 g the percent weight reduction of the eps was determined from these measurements and an average of 4 samples was calculated two controls per experimental variation were used the first controls contained all components of the experimental test tubes save for the microbial cultures and served to demonstrate that the polystyrene did not degrade in sterile lb the second controls contained all components of the experimental test tubes except that the microbial cultures were replaced by e coli to demonstrate that the assimilation of the lb by microorganisms did not cause a reduction of the mass of the test tubes the test tubes used were not hermetically sealed allowing gas loss which could cause weight loss and were manipulated with gloves to not leave grease prints every test tube in phases ii and iii had the same amount of medium 15 ml and eps approximately 0.5 g phase i isolation and selection soil sampling soil was sampled from the banks of the st-lawrence river in quebec in an area that was contaminated and in contact with eps this soil taken from the surface was used as a source of microorganisms that already had eps in their environment and were thus more likely to be able to degrade the polymer isolation of microorganisms by enrichment procedure ten grams of the soil sample were mixed in a sterile 500 ml glass container with 100 ml of lb 10 g of ground to increase the surface area eps and 250 ml of distilled water and then incubated at 27ºc for 7 days optimal growth conditions for soil microorganisms [16 following the incubation period 10 ml of the broth culture were transferred to another container with the same composition as the previous mixture the culture was incubated for the same length of time and the same temperature as previously this procedure was repeated 4 more times and the final solution was filtered 3 times using the gravity filtration method to remove the soil particles the goal of this enrichment procedure was to increase the chance of isolating microorganisms capable of degrading and adapting to eps half a gram 0.5 g of unbroken eps was placed in the final filtered solution and incubated at 27ºc for a period of 2 weeks which corresponds to the minimal length of time needed for industrial bioremediation [16 after the incubation period and weighting of the solution a mass reduction of 4.6 of the eps was observed this degradation was hypothesized to be caused by the microorganisms in the solution 5

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identification of the microorganisms in the final filtered broth culture dilutions 10 ppm of the final culture were streak plated to isolate individual colonies [17 three different strains were isolated and observed under a microscope and biochemical tests [annex 1 were conducted to identify the microorganism strains the three identified strains in the solution were streptomyces griseus pseudomonas putida and pseudomonas fluorescens which are known to be nonpathogenic [18 separate selection for polystyrene degrading microorganisms each individual strain was inoculated in a test tube containing 15 ml of lb and 0.5 g of ground eps each test tube was incubated at 28ºc for 7 days optimal growth condition specifically for the 3 microorganisms [18 after incubation 1 ml of the culture was transferred to another test tube containing 14 ml of lb and 1 g of ground eps each test tube was incubated in the same conditions as previously this procedure was repeated 8 more times for a total of 10 weeks each week the amount of eps was increased by 0.5 g and the concentration of lb was diluted by 5 with distilled water this method was done in order to induce eps adaptation of the three strains in order for them to be able to use the polymer as a carbon source by producing specific degradation enzymes phase ii biodegradation effect of lb concentrations on the biodegradation of eps according to microbial strains n=4 about 4.5x104 microorganisms/ml based on preliminary cultures that were replicated in triplicate in order to determine the growth curve over 2 weeks of each isolated and adapted strain were added to a test tube containing 0.5 g of unbroken eps and 15 ml of various concentrations of lb diluted with distilled water 0 lb 25 50 100 the cultures were incubated at a temperature of 28ºc for a period of two weeks the weight of the test tubes degradation of the eps and microbial concentrations were measured and quantified every two days the manipulations were done in triplicate and then were repeated on the next day n=4 this was done to compensate for fluctuations in atmospheric pressure and ambient humidity cultures were constantly aerated by continuous shaking at 150 rpm 6

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phase iii optimization optimization of biodegradation by combinations of microbial strains n=4 the three strains of microorganisms were hypothesized to have a strong relationship between them because they were present together in the final filtered solution in phase i different strain combinations were designed with a starting ratio of number of microorganisms of 1:1:1 maintaining a total of approximately 4.5x104 microorganisms/ml in the test tubes the test tubes were incubated at 28ºc with shaking for a period of two weeks microbial counting to count the number of microorganisms per milliliter of solution 1 ml of solution was sampled from the test tubes the solution was then diluted to 0.1 ppm and streaked onto lb agar plates per l tryptone 10 g yeast extract 5 g nacl 10 g 15 g agar with a sterilized nichrome inoculating loop after 48 hours of incubation at 28ºc the number of colonies was counted the number of individual colonies was multiplied by the dilution factor to obtain the number of live bacteria per ml in the solution optimization of biodegradation by varying lb concentrations and ph n=4 because the initial intervals of concentrations of liquid medium in the test tubes were very broad 25 intervals the optimal concentration of lb for eps biodegradation in two weeks was narrowed down to increasingly smaller intervals 5 then 1 0.2 and finally 0.1 intervals once the optimal lb concentration had been determined the ph of the media was also optimized using hcl or naoh to determine the optimal ph for degradation at intervals of 1 then 0.2 and finally 0.1 tris buffer 50 mm was used to keep the ph stable measurement of toxicity of eps-biodegrading cultures n=4 the toxicity of the optimized cultured solutions was determined by using the microtox® test model 500 with the nvn 6516 method [19 one milliliter from each experimental and control test tube was tested for toxicity every 2 days for 2 weeks categories of toxicity were determined using the tu20 value intensity of toxicity and comparing them to the nvn 6516 microbics toxicity values shown in table 1 table 1 standard tu20 values for water toxicity classification with microtox® test [19 category 1 2 3 4 5 tu20 0 2.2 2.2-10 11-100 100 toxicity non toxic slightly toxic toxic very toxic extremely toxic 7

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phase iv post-biodegradation test qualitative evaluation of the physical properties of treated and control eps n=4 the eps that was optimally treated for two weeks and the control for this degradation were individually heated in a pot of water the temperature at which there was a apparent change in the physical properties of the eps was noted by a third-party observer who was not aware which eps was the control and which group had undergone treatment the change in physical properties was identifiable when the eps became slightly sticky 3 results effect of lb concentrations phase ii results from the biodegradation in 2 weeks of weight reduction in phase ii are summarized in table 2 where the experimental test tubes exp are compared to their respective controls ctl every control for each experimental test tube did not have weight reduction the concentration of 25 lb was found to be the most effective concentration and 100 was the least effective for eps biodegradation p fluorescens was found to be the most effective microorganism and s griseus the least effective for eps biodegradation table 2 effect of lb concentrations on eps weight reduction for the three microbial strains after 2 weeks of treatment n=4 microorganisms lb 0 lb exp ctl streptomyces griseus pseudomonas fluorescens pseudomonas putida 0 3.2 ± 0.9 0 0 0 0 25 lb exp 0.08 ± 0.01 32.7 ± 2.5 4.9 ± 1.2 ctl 0 0 0 50 lb exp 0.03 ± 0.01 25.4 ± 1.6 2.1 ± 0.4 75 lb ctl 0 0 0 100 lb exp 0 2.3 ± 0.7 0 ctl 0 0 0 ctl exp 0 0 0 0 15.1 ± 2.6 0.4 ± 0.02 effect of combinations of different microorganisms phase iii results from the effect of the various combinations of microorganisms on eps biodegradation weight reduction along with the controls are shown in table 3 as in phase ii the concentration of 25 lb is shown to be the most effective and 100 lb the least effective for eps biodegradation 8

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the combinations of the three microorganisms s griseus p putida and p fluorescens resulted in the highest biodegradation percentage also the percent weight reduction was always higher when p fluorescens is present in the test tubes the effect of lb concentration on percent weight change of eps over two weeks at intervals of two days is graphically represented in figure 2 the cultures contained a mixture of s griseus p putida and p fluorescens all the controls are regrouped in one curve for better visual analysis the three strains were all still present at the end of the 2-week treatment table 3 effect of various combinations of strains on eps weight reduction at various lb concentrations after 2 weeks of treatment n=4 microorganisms lb 0 lb exp ctl 0 0 0 0 25 lb exp 56.5 ± 3.6 44.3 ± 3.1 1.4 ± 0.1 31.1 ± 2.4 50 lb 75 lb ctl 0 0 0 0 100 lb exp 10.7 ± 2.4 5.1 ± 0.7 0.3 ± 0.09 5.6 ± 1.2 ctl 0 0 0 0 ctl exp 0 0 0 0 34.5 ± 3.1 29.6 ± 2.4 0.8 ± 0.2 23.4 ± 2.6 ctl exp 0 0 0 0 22.3 ± 2.6 18.4 ± 1.6 0.3 ± 0.1 16.5 ± 1.4 10.3 ± 0.5 8.6 ± s griseus p fluorescens 0.9 0.7 ± s griseus p putida 0.2 7.5 ± p putida p fluorescens 0.6 s griseus p putida p fluorescens weight 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 0 lb 25 lb 50 lb 75 lb 100 lb controls time days figure 2 effect of lb concentration on eps weight reduction over 14 days using the combination of s griseus p putida and p fluorescens n=4 9

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effect of the variations of lb concentrations and ph using a combination of s griseus p putida and p fluorescens phase iii and post-degradation test phase iv the effect of the optimal variation of lb concentration is shown in table 4 this percentage of lb was determined by dividing the interval between 0 and 25 because 25 was the best value with intervals of 5 then once the two highest values were found to be 10 and 15 the interval between these two values was separated by intervals of 1 between 11.0 to 12.0 with intervals of 0.2 and finally between 11.4 and 11.6 with intervals of 0.1 thus the concentration of 11.5 lb resulted in the most effective concentration for eps biodegradation weight reduction of 64.6 in 2 weeks the effect of the ph on eps biodegradation in 11.5 lb is shown in table 5 the ph of 7.8 is shown to have the highest percentage of eps biodegradation weight reduction of 69.5 after 2 weeks the optimal ph was determined by varying intervals between 1 and 10 with steps of 1 then 7.0 to 8.0 with steps of 0.2 and finally 7.8 to 8.0 with steps of 0.1 further results are graphically shown in figure 3 about the biodegradation of eps with the combination of the three strains according to the optimal lb concentration and optimal ph over 2 weeks figure 4 shows the growth curve of the combination over the same length of time the most effective interval of eps biodegradation in each table is highlighted the eps biodegradation rate and microbial concentration are both shown to increase rapidly on day 4 table 6 shows the results of the post-degradation test table 4 effect of lb concentration on weight reduction of eps after 2 weeks n=4 lb concentrations weight reduction 11.4 lb exp ctl 62.5 ± 4.3 0 11.5 lb exp ctl 64.6 ± 3.2 0 11.6 lb exp ctl 63.4 ± 3.8 0 table 5 effect of ph on weight reduction of eps after 2 weeks in 11.5 lb n=4 ph exp weight reduction 69.5 ± 4.5 7.8 ctl 0 exp 68.6 ± 3.7 7.9 ctl 0 exp 67.2 ± 5.1 8.0 ctl 0 table 6 temperature of physical changes in treated and controlled eps after 2 weeks n=4 combination/temperature s griseus p putida p fluorescens 10 t°exp °c 97.8 ± 4.5 t°ctl °c 40.7 ± 3.2

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weight 100 80 60 40 20 0 0 2 4 6 8 10 12 14 time days s griseus p putida p fluorescens controls figure 3 eps weight reduction over 14 days under optimal conditions 11.5 lb ph 7.8 with the combination of s griseus p putida and p fluorescens n=4 microbial concentration microorganisms/ml 16000000 14000000 12000000 10000000 8000000 6000000 4000000 2000000 0 0 2 4 6 8 10 12 14 s griseus p putida p fluorescens e coli time days figure 4 change in microbial concentration over 14 days using the combination of s griseus p putida and p fluorescens in 11.5 lb with 7.8 ph and eps the solutions containing eps being degraded at optimal conditions with all three microorganisms were toxic from day 3 to 10 as shown in figure 5 tu20 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 12 14 experimentals controls time days figure 5 toxicity microtox® test in experimental and controlled test tubes over 14 days with the combination of s griseus p putida and p fluorescens in 11.5 lb with 7.8 ph 11

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4 discussion effect of lb concentrations phase ii according to individual strains as seen to table 2 p fluorescens is the only strain capable of efficiently degrading eps with an average weight reduction of 32.7 ± 2.5 in 25 lb the reason why 100 lb was the least effective concentration is because the microorganisms ignored eps as a carbon source in the presence of concentrated lb the two other strains s griseus and p putida were unable to degrade eps effectively no matter the concentrations of lb which suggests that p fluorescens is the only strain capable of producing extracellular degradation enzymes since the controls did not have weight reduction after 2 weeks degradation was exclusively due to microbial action optimization of eps biodegradation by combinations of strains the highest weight reduction of eps was achieved by combining the three microbes together with an average reduction of 56.5 ± 3.6 in 2 weeks in 25 lb every combination containing the strain p fluorescens had a high percentage of degradation compared to the combinations without the strain table 3 this supports the hypothesis that eps degrading enzymes are produced by p fluorescens furthermore the sum of the individual weight reduction percentages in 25 lb is inferior to the weight reduction of the combination of the three microorganisms in 25 lb tables 2 3 this result suggests that microbial synergy occurred in the test tubes which could explain the high percentage of weight reduction when the three strains were combined together in 25 lb an explanation for the low percentage of biodegradation by the microbes s griseus and p putida tables 2 3 is that these strains produce biosurfactants [20 biosurfactants are chemically active compounds that were shown to be produced by p putida [20 which can emulsify hydrocarbons [20 resulting in an augmentation of the surface area of eps thus the enzymes produced by p fluorescens are more likely to be effective when biosurfactants are in contact with eps which may explain the microbial synergy and the high percentage of biodegradation when organisms are combined optimization of eps biodegradation by variations of lb concentrations and ph optimal biodegradation was obtained with 11.5 lb with a ph of 7.8 for an average weight reduction of 69.5 ± 4.5 in 2 weeks table 5 although the result at 11.5 lb was not statistically different from results at 11.4 and 11.6 lb t-test comparing treatments p<0.05 it is significantly greater than the percentages of weight reduction in 25 lb in table 3 p=0.00021 which suggests that optimization of eps biodegradation was successful 12

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because the eps biodegradation and microbial concentration increase rapidly on day 4 this suggests that the three strains are not only capable of degrading eps optimally but are also capable of metabolizing the polymer in order to increase microbial growth no indication of breakdown of eps was detected in the control treatments despite signs of microbial growth figures 3 4 analysis of toxicity microtox® assays were done to measure the toxicity in the test tubes the liquid environment in the experimental test tubes on days 4 6 and 8 was toxic which suggest that the enzymes along with the biosurfactants were able to degrade eps into the toxic compounds styrene and bisphenol a the reduction after day 5 is a result of the metabolizing of these toxic chemicals by p putida and p fluorescens past research shows that p putida and p fluorescens are capable of rapidly metabolizing these chemical compounds [21 22 thus this method could be used to reduce the toxicity of partially degraded eps-contaminated water effect of the enrichment procedure the enrichment procedure developed in this experiment demonstrates that it is possible to select for microorganisms capable of degrading certain types of polymers in this case eps without this selection pressure eps biodegradation would have been limited 4.6 of weight reduction before the enrichment procedure compared to 69.5 after enrichment and optimization practical and industrial applications the eps biodegradation method in this experiment could be easily industrially applied to treat eps-contaminated water with the three isolated and adapted microorganisms now that the optimal conditions have been determined hence it could be possible to isolate the enzymes and biosurfactants produced by the three microorganisms to degrade eps in water or before it ends up in rivers or oceans because the eps exposed to the microbes was shown to have a significantly lower temperature of physical change table 6 this suggests that partially degraded eps could facilitate gasification since treated eps is less resistant to heat identification of the enzymes and biosurfactants is the next step in the project 5 conclusions 1 the biotechnological procedure developed in this experiment successfully achieved a high percentage of eps degradation and a high reduction of toxicity in a liquid environment 2 it is possible to apply this biodegradation method to reduce the amount of eps in rivers and oceans thus saving the marine environment 13

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3 it is possible to apply this biodegradation method to reduce the toxicity of eps contaminated water 4 this method can potentially facilitate current eps degradation methods it is also an inexpensive environmentally friendly and is a potential efficient alternative to get rid of eps from water 6 references [1 sri consulting http sriconsulting.com/wp/public/reports/polystyrene xml=http 10.2.0.53/cgibin/texis/webinator/search/pdfhi.txt?query=polystyrene&pr=super&prox=page&rorder=500&rprox=500 &rdfreq=500&rwfreq=500&rlead=500&sufs=0&order=r&d=4c0a7a5e7 accessed feb 2010 [2 myers lr 2007 the 100 most important chemical compounds pp 267 greenwood publishing group westport ct [3 polytechnique montréal opération le vert tasse le verre [operation green gets rid of the cups http www.polymtl.ca/enviropoly/operationvtv/verre.php accessed nov 2009 [4 american chemical society http portal.acs.org:80/portal/acs/corg/content nfpb=true pagelabel=pp_articlemain&node_id =222&content_id=cnbp_024352&use_sec=true&sec_url_var=region1 uuid=f26a9407-2c78-475c8b07-3ceb12388611 accessed apr 2010 [5 american chemical society http portal.acs.org:80/portal/acs/corg/content nfpb=true pagelabel=pp_articlemain&node_id =222&content_id=cnbp_022763&use_sec=true&sec_url_var=region1 uuid=866d6f03-a685-484baf53-c6bc445d4872 accessed sept 2009 [6 greenpeace http www.greenpeace.org/international/campaigns/oceans/pollution/trash-vortex accessed dec 2009 [7 material safety data sheet http www.wind-lock.com/dsn/wwwwindlockcom/content/pdf/eifs/epsmsdsforfoamcuttinggrade54.pdf pp 8 accessed jan 2010 [8 weyer p riley d 2001 endocrine disruptors and pharmaceuticals in drinking water pp 89-91 american water works association iowa city ia [9]sirc.http www.styrene.org/news_styrene_human_health/sirc_statement_styrene_iarc.html accessed jan 2010 [10 free drinking water http www.freedrinkingwater.com/water-contamination/styrenecontaminants-removal-water.htm accessed jan 2010 14

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