OUTREACH

Alan Scarlett

ProjectOUTREACHProf. Steve RowlandDr Charles WestDave JonesCollaborative research in JapanProf. Atsushi Ishimatsu and Dr Awantha Dissanayake

In Japanese: Watashi wa Alan des. Watashi wa Igirisu-jin-des. I am Alan, Im from England. I speak a little Japanese but Im not very good yet. I now will speak English Ima kara eigo o ha-na-shi-mas. My apologies for my poor pronunciation. Im sure your English is much better than my Japanese. Today I will tell you a little bit about where Im from, my research group and the current project Im working on. And of course, why I am in Japan.1

Location of University of Plymouth

Plymouth50 kmI am from Plymouth in the SW of the UK2Plymouth between 2 rivers

250,000 population30,000 studentsPlymouth is situated at the mouth of 2 rivers, the Tamar and the Plym. It has a population of about a quarter of a million people and the university has about 30 000 students3Centre for Research in BiogeochemistryBiogeochemistry and Environmental Analytical Chemistry (BEACh) group Petroleum and Environmental Geochemistry Group (PEGG)Inorganic chemistry e.g. nutrients, metals and radionuclideshttp://www.plymouth.ac.uk/research/beachOrganic chemistryhttp://www.research.plymouth.ac.uk/pegg/I am part of the centre for biogeochemistry research. This comprises of 2 groups. The beach group has an excellent reputation for research into the fate and impact of nutrients, metals and radionuclides in the environment. And my group, Petroleum and environmental geochemistry group is mainly concerned with organic chemistry and Im to going say a bit more about what my group is interested in.4Climate change- Sea ice proxy

Pharmaceuticals in the environment

Unresolved complex mixtures within weathered oilsChemical structuresPEGG - what we do:So our research covers climate change, in particular using chemicals produced from diatoms as a proxy for sea ice cover in the past. We are also interested in pharmaceuticals in the environment. Another area of research we have been involved in for many years is trying to find out for is in unresolved complex mixtures found within weathered oils. So what have all these areas of research got in common? Chemical structures. It is remarkable how similar many of these structure are. My main area of research is concerned with the toxicity of the compounds found within these unresolved complex mixtures5Petroleum and Environmental Geochemistry Group (PEGG)

OUTREACH teamHere is my research group, : Prof Steve Rowland, Dr Charles West and our PhD student Dave Jones. Actually this is a bit out of date, we are missing a few people6Team Outreach

Whats all this stuff contaminating the seas?How much is there?Is it toxic?Where in the world?This is what we look like would look like if we lived in South park. The questions our project are trying to answer are: .....7OUTREACH - rationaleMarine pollution by organic chemicalsWorldwide regulations (e.g. REACH, USEPA)Priority pollutants (e.g. polycyclic aromatic hydrocarbons, PAH)Based on known (or suspected) detrimental biological activity (e.g. carcinogenicity)

So the project I am involved in is called outreach and is concerned with marine pollution by organic pollutants. Worldwide environmental regulators such as the USEPA and a new directive in Europe called REACH are only concerned with particular contaminants such as priority pollutants but these are only a small fraction of chemicals that are in the environment8OUTREACH project5 year study funded by European Research CouncilComposition and toxicity of overlooked contaminantsPolar unresolved complex mixtures from oilsExtract water soluble fraction oilsAnalyse by state-of-the-art instruments Synthesise and test candidate compounds toxic effectsCollect samples from sites around the worldAim to inform environmental legislation

Our study is funded by the european research council for 5 y. So we will try to indentify toxic compounds that have been overlooked by the regulators. We have a new very sophisticated analytical instrument to help us and we have the capacity to synthesise candidate compounds so we can test them for toxic effects. Hopefully, this will then inform the legislators and make the marine environment a safer place9Oil a very complex mixture

Many people tend to think of oil as being a chemical but it is of course a very complex mixture of hundreds of thousands of compounds10Legislation focuses on priority pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs)

TimeResponsee.g. phenanthreneselected ion monitored m/z 178 Analysed by gas chromatography mass spectrometry (GC-MS)OUTREACH project

When compounds from oil get analysed by standard gas chromatography mass spectrometry methods, it is common to look for a particular compound. Here for example the 188 ion is being monitored in order to identify phenanthrene11But what if we look for all compounds present?

TimeResponseWe see that there are thousands of unknown compounds what if these are also toxic?Unresolved complex mixture (UCM)OUTREACH projectBut what if we look for all the compounds present? We now see that there are so many that they cannot be separated or resolved so this has been termed the unresolved complex mixture or UCM for short. What if this UCM contains toxic compounds that are being overlooked?12Comprehensive two-dimensional gas chromatography (GCxGC)Time-of-flight mass spectrometer (ToF-MS)

Normal gas chromatography - mass spectrometry (GC-MS)

D

13We now have a new analytical instrument to help separate and identify these compounds. In normal gas chromatography Time of Flight Mass Spectrometer (ToF-MS)Ion SourceReflectorDetector14Time of Flight Mass Spectrometer (ToF-MS)15GCxGC-TOF-MS

Time of Flight Mass Spectrometer(TOF-MS)Tandem Gas Chromatograph (GCxGC)Cryogenic coolant armSo this the actual instrument. As you can see it is quite small.16A complex mixture by GCxGC-ToF-MS

Time on 1st columnTime on 2nd columnAnd this is the sort of output we obtain. Each one of these peaks represents an individual compound for which we can obtain mass spectra. At the back of this picture you see what would have been obtained by normal Gas chromatography17Major aromatic compound groups 11.522.533.545001000150020002500Ist dimension retention time (s)2nd dimension retention time (s)Peaks = 2376Increasing volatilityIncreasing polaritym/zMolecular ionm/z 24680100120140160180200220240260020000400006000080000100000120000140000160000180000200000m/z-->Abundance7491105119246

OUTREACH project past researchThis is another way of looking at the results generated by the GCxGC. Along the horizontal axis is the time the compounds spent passing through the first column and on the vertical axis the time on the 2nd column. Each dot represents the tip of a peak and an individual compound. So we can click on a peak, look at the mass spec and try and identify what it is. This is an alkybenzene and we can see a whole band of these. Similarly we can see bands of alky indans and tetralins, and another band of alkyl naphthalenes etc. So now we are in position to test if these compounds are toxic.18

Mussels (Mytilus spp) as sentinal organisms for assessing health of marine communitiesTo test the toxicity of the compounds we usually use mussels as they bioaccumulate hydrophobic chemicals in their tissues and the also act as sentinal organisms for the general health of marine communities1900.511.522.533.5436086013601860236028603360386043601st Dimension RT (s)2nd Dimension RT (s)Contaminants in wild mussels

Total UCM 3440 PeaksAlkylbenzenes 742 Peaks

Apolar column retention time Polar column retention time Possible structure branched C12 alkylbenzene

Toxicity testingClearance rate assayTissue EC20 = 10 g/g

Booth et al. (2007) Environ. Sci. Technol; Scarlett et al. (2008) Environ. Toxicol. Chem

When we look at the compounds within the tissues of wild mussels from polluted sites we see these branched alkylbenzenes so we were able to purchase a mixture of these compounds and test their toxicity. From these tests we know that about 10 ug of alkylbenzenes in a gram of dry tissue will reduce the rate at which the mussels can filter algae by about 20%20

What if the mussels are eaten?

Mussels exposed to alkylbenzenesfor 48 hoursContaminated mussels fed to crabs for 7 daysStandardised behaviour tests coupled to physiological measurementsWhat if the mussels are eaten?

Behaviour of shore crab Carcinus maenasMussel tissue concentration consistent with realistic UK marine waters contamination

Scarlett et al, (2009). Environ. Toxicol. & Chem. But what happens if another animal eats mussels contaminated by the branched alkylbenzenes. Lets watch this crab. It is been fed for a week on mussels contaminated by an enviromentally realistic concentration. A healthy crab would by now have dashed over, grabbed the cockle, orientated it in a particular way and broken through the shell but this one is struggling. . 22

Branched indans & tetralinsSynthesis by Grignard reaction Multiple isomers

Synthesised compounds

Booth et al. (2008) Environ. Sci. Technol.Toxicity testingClearance rate assayTissue EC20 = 13 g/g

So thats the alkylbenzenes but what if you cant buy the chemicals of interest? We synthesise them. These are some branched alkyl indans and tetralins that we saw earlier. Thanks to our ability to synthesise the compounds we have been able to test their toxicity and now know that they have similar toxicity as the branched alkylbenzenes when based on their tissue concentration.Lunch?

Many other alkylated compounds present in mussel tissuesHigher organisms metabolise compoundsNo evidence for biomagnificationBut food for thought.So that is just a few of compounds in the unresolved complex mixtures found in mussels. There are many others. Higher organisms such as ourselves can metabolise these compounds so we have no evidence that these compounds can biomagnify through the food web but it does make you think.24Polar complex mixtures

Water soluble fraction of oil (inc. sulphur, nitrogen and oxygenated compounds)Produced waters oil rigsoil sands extractionVery complexToxic?Hormonal effects?

Melbye et al, (2009). Environ. Toxicol & Chem.

Our current project is concerned with the more water soluble fraction of oils. These compounds may contain sulphur or nitrogen or oxygen. They are found in produced waters such as from oil rigs and from oil sands extraction. These are very very complex mixtures but they contain toxic components some of which have effect the homonal activity of organisms25

Current research:Athabasca oil sands, Alberta Canada

One area of current research is waste waters produced by the extraction of oil sands26

This is a huge industry in Canada and covers an enormous area. Here is the island of Kyushu on the same scale. You can see that the oils sands industry covers a greater area this entire island

27

Acids within Oil Sands Produced WatersFrom Alberta CanadaInternal standardUCMDespite being extremely complex we have been able to separate many of the compounds and, for the first time ever, identify compounds within the mixture. We have several papers under review describing this major breakthrough. Once identified we can test their toxicity. We have already synthesised and tested quite a few and are currently investigating many more.28OUTREACHThousands of compounds within unresolved complex mixtures (UCMs) ignoredCompound 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?So that is the outreach project. Thousands of potentially toxic compounds have been ignored but we are beginning to make advances. We have already identifies lots of toxic hydrocarbons and are now looking at the polar compounds. Another question is of course how much of these compounds are waters and organisms around the world?29The Japan connectionFunded by the Japanese Society of the Promotion of Science (JSPS) So now we come onto the Japanese connection. What am I doing here?30

Halpern et al (2008) A Global Map of Human Impact on Marine Ecosystems . Science, 319

Japanese waters amongst highest impactedGlobal map of cumulative human impact across 20 ocean ecosystem types A couple of years ago, a paper in Science identified regions of the worldthat had been particulary impacted by human activity. My own backyard around the UK coast was one area but the sea around Japan were also highlighted as highly affected31Health effects assessment (1)Mussel clearance rateBiomarkers e.g.PhysiologicalCellularMembrane integrity (neutral red assay)Immune response (phagocytosis assay)

In order to test the health of the marine environment, Ive shipped over this instrument so I can carry out the mussel clearance rate assay. With the biomarker expert, Awantha, we will also look at various cellular biomarkers such as membrane integrity and immune function32Health effects assessment (2)SeagrassPhotosynthetic efficiency

Assessed using Plant Efficiency AnalyserFast fluorescence Max efficiency of PSII (Fv/Fm)Pool size of electron receptors (strongly affected by PSII inhibitors e.g. triazine herbicidesVitality (Performance Index)Scarlett et al, (1997, 1999) Mar. Pollut. Bull; Scarlett et al, (1999) Aquat. Toxicol. We will also use seagrasses as passive sampling devices and we can assess their health in situ by measuring their photosynthetic efficiency33Water sampling using passive samplers

Polar Organic Chemical Integrative Sampler (POCIS)28 day deploymentAccumulates low- mid value log Kow compoundsPharmaceuticals and pesticides I have also brought some chemical based passive samplers that are specifically designed to accumulate polar compounds.34Tissue & water analysesShip back to PlymouthExtract tissues and POCISAnalyse by GCGC-TOF-MSIdentify compoundsQuantify contaminants

So the bioassays and biomarkers will be conducted here and then the water and tissue samples will be shipped back to Plymouth for chemical extraction and analyses by our new GXxGC35Where to collect samples?

At this point as we reach the end of the talk it is normal for the audience to ask the sspeaker questions. However, today I want to ask you for you advice on where to collect mussels or oysters and where to locate the passive sampling devices.36

Fig. 1. Global map (A) of cumulative human impact across 20 ocean ecosystem types.
B S Halpern et al. Science 2008;319:948-952
Published by AAAS

Global map (A) of cumulative human impact across 20 ocean ecosystem types. (Insets) Highly impacted regions in the Eastern Caribbean (B), the North Sea (C), and the Japanese waters (D) and one of the least impacted regions, in northern Australia and the Torres Strait (E).