! Polycyclic Aromatic Hydrocarbons in Mussel from Iceland Food safety and environmental aspect Berglind Ósk Alfreðsdóttir Thesis for the degree of Master of Science Food Science and Nutrition School of Health Science ! ! Polycyclic Aromatic Hydrocarbons in mussel from Iceland ! Food safety and environmental aspect Berglind Ósk Alfreðsdóttir Thesis for the degree of Master of Science Supervisors: Helga Gunnlaugsdóttir and Hrönn Jörundsdóttir Food Science and Nutrition School of Health Science University of Iceland October 2014 ! ! ! ! This thesis is for Master degree in Food Science and may not be copied in any form without the permission of the rights holder ! ! ! ABSTRACT ! Polycyclic aromatic hydrocarbons (PAHs) are a substance of great environmental and health concern as they can find their way to the human food chain through the ocean’s ecosystem. PAHs are a group of compounds that are composed of fused aromatic rings formed through the combustion of organic matter. The US Environmental Protection Agency (US-EPA) has identified 16 PAHs of particular importance due to their toxicity as priority pollutants. These are the PAHs most commonly used in monitoring ! projects to gather information and assess the status of food products regarding undesirable substances. The aim of this master thesis was to investigate temporal trends of PAHs in the Icelandic coastal environment in the timeframe 1992-2011. The importance of monitoring the PAHs is twofold: with regard to food safety since they can have mutagenic/carcinogenic effects on humans and from the environmental aspect because they can provide evidence of human activity in the area. Three locations, Brekka in Mjóifjörður, Úlfsá in Skutulsfjörður and Hvassahraun in Hafnarfjörður, were chosen as investigation sites and blue mussels (Mytilus edulis) were chosen as a monitoring species. Extraction of PAHs in the blue mussels were performed by using microwave- assisted extraction followed by solid phase extraction clean up. Identification and quantification was performed with gas chromatograph coupled to a mass spectrometer (GC-MS). In this thesis, the sum of the concentration of the 16 PAHs is expressed as Σ16 . The PAH results showed that the concentration in Hvassahraun is relatively low and stable during the time period, with a maximum of Σ16 concentration 345 ng/g d.w in the PAH year 1997. In Úlfsá, the concentration was also generally low and stable during the time period, with a maximum of Σ16 concentration of 264 ng/g d.w. collected in PAH 2000. The temporal trend in Mjóifjörður showed an increase in PAHs concentration during the time period. The concentration increased in the year 2000, and again between 2007 and 2010, with the highest value of 1532 ng/g d.w. obtained in 2010. The high concentration measured in Mjóifjörður could be due to increased human activity in the fjord due to fish farming in this period. The results indicate that the Icelandic coastline environment is ideal for utilization for food production. iii! ! ! ÁGRIP Fjölhringa arómatísk vetniskolefni (PAH efni) eru efnasambönd sem geta komist í fæðukeðju manna og haft skaðleg áhrif á lýðheilsu. PAH efni er flokkur efna úr arómatískum kolefnum sem myndast við bruna lífrænna efna, bæði af mannavöldum og með náttúrulegum hætti. Umhverfisstofnun Bandaríkjanna (EPA) hefur valið sextán PAH forgangsefni sem eru talin sérstaklega skaðleg heilsu manna og umhverfinu. Fyrir þessum sextán PAH forgangsefnum er skimað í umhverfisvöktun til þess að meta ! magn óæskilegra PAH efna, meðal annars í matvælum. Markmið verkefnisins var að mæla magn PAH efna í kræklingi (Mytilus edulis) og skoða þróun þeirra við strandlengju Íslands á árunum 1992-2011. Skoðuð voru sýni frá þremur stöðum, frá Hvassahrauni í Hafnarfirði, Úlfsá í Skutulsfirði og Brekku í Mjóafirði, sem áður höfðu verið notuð í mengunarvöktun lífríkis sjávar við Ísland. Í efnagreiningunum var notaður úrdráttur með örbylgjum og kísilgelsúlur til þess að hreinsa sýnin. Að lokum voru þau aðgreind og magngreind með GC-MS. Í ritgerðinni er Σ16 skilgreind sem summa PAH forgangsefnanna sextán. PAH Niðurstöðurnar sýna að styrkur þeirra í Hvassahrauni er tiltölulega lágur og stöðugur yfir tímabilið en hæsta gildi Σ16 mældist 345 ng/g á þurrvigt í sýni frá 1997. Í PAH Úlfsá var styrkurinn einnig tiltölulega lágur og stöðugur en hæsta gildi Σ16 mældist PAH 264 ng/g þurrvigt í sýni frá árinu 2000. Í Mjóafirði eykst styrkur PAH efnanna á tímabilinu. Styrkurinn jókst árið 2000 og svo aftur árin 2007-2010 frá tiltölulega stöðugu bakgrunnsgildi. Hæsta gildi Σ16 mældist 1532 ng/g þurrvigt árið 2010. PAH Líklega má rekja hækkunina í Mjóafirði til aukinna umsvifa í firðinum í tengslum við fiskeldi. Niðurstöður verkefnisins gefa vísbendingu um að íslenskt strandsvæði sé tiltölulega hreint með tilliti til PAH-efna og því kjörið til nýtingar á sjávarfangi og annarra lífvera sem lifa við strendurnar. iv! ! ! ACKNOWLEDGEMENTS This study was conducted at Matís ohf Reykjavík, which also financed the project. I would like to thank my supervisors Helga Gunnlaugsdóttir and Hrönn Jörundsdóttir and the committee member Þórhallur Ingi Halldórsson for their knowledge and guidance through the study. I also want to send my sincere thanks to Vordís Baldursdóttir for carrying out the GC-MS analysis of the samples and her guidance ! regarding the analysis of the results from the GC-MS. Lastly I want to thank my family for their support throughout my studies. v! !! ! TABLE OF CONTENTS ! ABSTRACT .................................................................................................................. iii ÁGRIP ........................................................................................................................... iv ACKNOWLEDGEMENTS ......................................................................................... v TABLE OF CONTENTS ............................................................................................ vi LIST OF FIGURES ................................................................................................... viii LIST OF TABLES ....................................................................................................... ix !ABBREVIATIONS ....................................................................................................... x 1. INTRODUCTION ..................................................................................................... 1 2. LITERATURE REVIEW ........................................................................................ 2 2.1 Chemical and physical characteristics of PAHs ................................................... 2 2.1.1 Definition ....................................................................................................... 2 2.1.2 Formation ....................................................................................................... 3 2.1.3 Classification .................................................................................................. 4 2.1.4 Characteristics ................................................................................................ 4 2.2 Sources and exposure of PAHs ............................................................................. 5 2.2.1 Environmental concerns regarding PAHs ...................................................... 5 2.2.2 PAHs in the marine environment ................................................................... 6 2.2.3 PAHs in food .................................................................................................. 7 2.2.4 Exposure in humans ....................................................................................... 7 2.2.5 PAHs in drinking water ................................................................................. 9 2.3 Kinetics and metabolism in humans ..................................................................... 9 2.3.1 Absorption of PAHs ....................................................................................... 9 2.3.2 Distribution and excretion of PAHs ............................................................. 10 2.3.3 Metabolism of PAHs .................................................................................... 10 2.4 Health effects ...................................................................................................... 11 2.4.1 Health effects in children ............................................................................. 12 2.5 Conventions and regulations ............................................................................... 12 2.5.1 Conventions ................................................................................................. 12 2.5.2 Regulations .................................................................................................. 13 2.6 Blue mussel as biomarker ................................................................................... 13 2.7 Analytical methods for determination of PAHs .................................................. 15 2.7.1 Microwave-assisted extraction ..................................................................... 15 2.7.2 Solid phase extraction .................................................................................. 16 2.7.3 GC-MS ......................................................................................................... 16 2.9 Research objectives ............................................................................................. 17 3. MATERIAL AND METHODS ............................................................................. 18 3.1 PAHs determination in mussel ............................................................................ 18 3.1.1 Samples and samples sites ........................................................................... 18 3.1.2 Chemicals ..................................................................................................... 19 3.1.3 Microwave-assisted extraction ..................................................................... 20 3.1.4 Filtration and solid phase extraction ............................................................ 20 3.1.5 GC-MS ......................................................................................................... 21 3.1.6 Quality assurance ......................................................................................... 22 3.2.7 Calculations of the PAHs compounds ......................................................... 22 vi! ! ! 4. RESULTS ................................................................................................................ 24 4.1 Hvassahraun sample site ..................................................................................... 25 4.2 Úlfsá sample site ................................................................................................. 28 4.3 Mjóifjörður sample site ....................................................................................... 30 4.4 Reference sample and recovery .......................................................................... 33 5. DISCUSSION .......................................................................................................... 34 5.1 PAHs concentration at the sample site in Hvassahraun ...................................... 34 5.2 PAHs concentration in Úlfsá .............................................................................. 35 5.3 PAHs concentration in Mjóifjörður .................................................................... 35 5.4 Comparison between the three locations ............................................................ 37 5.5 Origin of the PAHs ............................................................................................. 38 ! 5.6 PAHs concentration from the Arctic and sub-Arctic parts of the North Atlantic Ocean ........................................................................................................................ 39 5.7 PAHs analytical method ..................................................................................... 41 6. CONCLUSIONS ..................................................................................................... 42 7. REFERENCES ........................................................................................................ 43 vii! ! ! LIST OF FIGURES ! Figure 1: The sixteen priority PAHs .............................................................................. 3 Figure 2: Blue mussel (Mylitus edulis) (Brey, 2008) ................................................... 14 Figure 3: Map showing the sampling locations in Iceland of blue mussels used in the present study for PAHs analyses .......................................................................... 19 Figure 4: a) Shows the solid phase extraction equipment b) Shows the nitrogen blowing equipment used in this present PAHs assessment study ....................... 21 ! Figure 5: Shimadzu GC-MS (QP2010 ultra) used in the present study for PAHs identification and quantification ......................................................................... 21 ! Figure 6: An example of five-point calibration curve for benzo[a]pyrene ................... 22 Figure 7: Temporal trends in PAHs concentration in blue mussel (Mylitus edulis) for Hvassahraun sample site, collected in 1992-2011. The figure shows upper and lower bound sum for the Σ16 in ng/g d.w. mussel sample .............................. 25 PAH Figure 8: Temporal trends in PAHs concentration in blue mussel (Mylitus edulis) for Úlfsá sample site, collected in 1997-2011. The figure shows upper and lower bound sum for the Σ16 in ng/g d.w. mussel sample ........................................ 28 PAH Figure 9: Temporal trends in PAHs concentration in blue mussel (Mylitus edulis) for Mjóifjörður sample site, collected in 1996-2011. The figure shows upper and lower bound sum for the Σ16 in ng/g d.w. mussel sample .............................. 30 PAH Figure 10: Comparison of the upper bound sum Σ16 concentration in blue mussel PAH (Mylitus edulis) collected in 1992-2011 for the three sampling locations, Hvassahraun, Úlfsá and Mjóifjörður ..................................................................... 38 Figure 11: Cross plots for the ratios of FluA/(FluA+Pyr) vs. AnT/(AnT+PhA). ......... 39 viii! ! ! LIST OF TABLES Table 1: Physical and chemical properties of the 16 PAHs in the present study ............ 5 ! Table 2: Total dietary exposure to benzo(a)pyrene (BaP) .............................................. 8 ! Table 3: Consumer exposure to Benzo(a)pyrene (BaP) and ........................................... 9 ! Table 4: Classification of carcinogenic PAHs (IARC, 2014) ....................................... 12 ! Table 5: Sample locations, coordinates, site specifications and descriptions of the ! locations ................................................................................................................ 19 ! Table 6: Concentration of analyzed PAHs in ng/g d.w. in blue mussel (Mytilus edulis) samples from Hvassahraun sample site 1992-2011 .............................................. 27 ! Table 7: Concentration of analyzed PAHs in ng/g d.w. in blue mussel (Mytilus edulis) samples from Úlfsá sample site 1997-2011 .......................................................... 29 ! Table 8: Concentration of analyzed PAHs in ng/g d.w. in blue mussel (Mytilus edulis) samples from Mjóifjörður sample site 1996-2011 ................................................ 32 ! Table 9: Average recovery and relative standard deviation for recovery ..................... 33 ix! !