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Project ‘OUTREACH’

Project ‘OUTREACH’. Alan Scarlett. Prof. Steve Rowland Dr Charles West Dave Jones. Collaborative research in Japan Prof. Atsushi Ishimatsu and Dr Awantha Dissanayake. Location of University of Plymouth. 50 km. Plymouth. Plymouth – between 2 rivers. 250,000 population 30,000 students.

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Project ‘OUTREACH’

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  1. Project‘OUTREACH’ Alan Scarlett Prof. Steve Rowland Dr Charles West Dave Jones Collaborative research in Japan Prof. Atsushi Ishimatsu and Dr AwanthaDissanayake

  2. Location of University of Plymouth 50 km Plymouth

  3. Plymouth – between 2 rivers 250,000 population 30,000 students

  4. Centre for Research in Biogeochemistry Biogeochemistry and Environmental Analytical Chemistry (BEACh) group Petroleum and Environmental Geochemistry Group (PEGG) Inorganic chemistry e.g. nutrients, metals and radionuclides http://www.plymouth.ac.uk/research/beach Organic chemistry http://www.research.plymouth.ac.uk/pegg/

  5. PEGG - what we do: Climate change - Sea ice proxy Pharmaceuticals in the environment Unresolved complex mixtures within weathered oils Chemical structures

  6. Petroleum and Environmental Geochemistry Group (PEGG) OUTREACH team

  7. What’s all this stuff contaminating the seas? How much is there? Team “Outreach” Where in the world? Is it toxic?

  8. OUTREACH - rationale • Marine pollution by organic chemicals • Worldwide regulations (e.g. ‘REACH’, USEPA) • Priority pollutants (e.g. polycyclic aromatic hydrocarbons, PAH) • Based on known (or suspected) detrimental biological activity (e.g. carcinogenicity)

  9. OUTREACH project • 5 year study funded by European Research Council • Composition and toxicity of ‘overlooked’ contaminants • Polar unresolved complex mixtures from oils • Extract water soluble fraction oils • Analyse by state-of-the-art instruments • Synthesise and test candidate compounds toxic effects • Collect samples from sites around the world • Aim to inform environmental legislation

  10. Oil – a very complex mixture

  11. OUTREACH project Legislation focuses on priority pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs) e.g. phenanthrene selected ion monitored m/z 178 Analysed by gas chromatography – mass spectrometry (GC-MS) Response Time

  12. OUTREACH project But what if we look for all compounds present? We see that there are thousands of unknown compounds – what if these are also toxic? Unresolved complex mixture (UCM) Response Time

  13. Comprehensive two-dimensional gas chromatography (GCxGC) • Time-of-flight mass spectrometer (ToF-MS) Normal gas chromatography - mass spectrometry (GC-MS) D

  14. Time of Flight Mass Spectrometer (ToF-MS) Detector Reflector Ion Source

  15. Time of Flight Mass Spectrometer (ToF-MS)

  16. GCxGC-TOF-MS Cryogenic coolant arm Time of Flight Mass Spectrometer (TOF-MS) Tandem Gas Chromatograph (GCxGC)

  17. A complex mixture by GCxGC-ToF-MS Time on 2nd column Time on 1st column

  18. Increasingpolarity Increasing volatility OUTREACH project – past research Major aromatic compound groups 4 Peaks = 2376 3.5 3 2nd dimension retention time (s) 2.5 2 1.5 1 500 1000 1500 2000 2500 st I dimension retention time (s) 119 200000 180000 Molecular ion m/z 246 160000 140000 105 120000 91 Abundance 100000 80000 60000 40000 74 246 20000 0 m/z--> 80 100 120 140 160 180 200 220 240 260 m/z

  19. Mussels (Mytilusspp) as sentinal organisms for assessing health of marine communities

  20. 4 3.5 3 2.5 2nd Dimension RT (s) 2 1.5 1 0.5 0 360 860 1360 1860 2360 2860 3360 3860 4360 1st Dimension RT (s) Contaminants in wild mussels Toxicity testing Clearance rate assay Tissue EC20 = 10 µg/g Polar column retention time Alkylbenzenes 742 Peaks Possible structure branched C12 alkylbenzene Total UCM 3440 Peaks Apolar column retention time Booth et al. (2007) Environ. Sci. Technol; Scarlett et al. (2008) Environ. Toxicol. Chem

  21. What if the mussels are eaten? Contaminated mussels fed to crabs for 7 days Standardised behaviour tests coupled to physiological measurements Mussels exposed to alkylbenzenes for 48 hours

  22. What if the mussels are eaten? • Behaviour of shore crab Carcinusmaenas • Mussel tissue concentration consistent with realistic UK marine waters contamination Scarlett et al, (2009). Environ. Toxicol. & Chem.

  23. Synthesised compounds Branched indans & tetralins • Synthesis by Grignard reaction • Multiple isomers Toxicity testing Clearance rate assay Tissue EC20 = 13 µg/g Booth et al. (2008) Environ. Sci. Technol.

  24. Lunch? • Many other alkylated compounds present in mussel tissues • Higher organisms metabolise compounds • No evidence for biomagnification • But food for thought….

  25. Polar complex mixtures • Water soluble fraction of oil (inc. sulphur, nitrogen and oxygenated compounds) • Produced waters • oil rigs • oil sands extraction • Very complex • Toxic? • Hormonal effects? Melbyeet al, (2009). Environ. Toxicol & Chem.

  26. Current research: • Athabascaoil sands, Alberta Canada

  27. Acids within Oil Sands Produced Waters From Alberta Canada Internal standard UCM

  28. OUTREACH • Thousands of compounds within unresolved complex mixtures (UCMs) ignored • Compound classes include hydrocarbons and more polars (e.g. carboxylic acids) • So OUTREACH asks: • what are these unresolved compounds? • how toxic are they? • how much - in various polluted regions of the world?

  29. The Japan connection Funded by the Japanese Society of the Promotion of Science (JSPS)

  30. Halpernet al (2008) A Global Map of Human Impact on Marine Ecosystems . Science, 319 Global map of cumulative human impact across 20 ocean ecosystem types Japanese waters amongst highest impacted

  31. Health effects assessment (1) Mussel clearance rate Biomarkers e.g. • Physiological • Cellular • Membrane integrity (neutral red assay) • Immune response (phagocytosis assay)

  32. Health effects assessment (2) Seagrass Photosynthetic efficiency • Assessed using ‘Plant Efficiency Analyser’ • Fast fluorescence • Max efficiency of PSII (Fv/Fm) • Pool size of electron receptors (strongly affected by PSII inhibitors e.g. triazine herbicides • Vitality (Performance Index) Scarlett et al, (1997, 1999) Mar. Pollut. Bull; Scarlett et al, (1999) Aquat. Toxicol.

  33. Water sampling using passive samplers • Polar Organic Chemical Integrative Sampler (POCIS) • 28 day deployment • Accumulates low- mid value log Kow compounds • Pharmaceuticals and pesticides

  34. Tissue & water analyses • Ship back to Plymouth • Extract tissues and POCIS • Analyse by GC×GC-TOF-MS • Identify compounds • Quantify contaminants

  35. Where to collect samples?

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