Liege 2017 – PCBs, PBDEs and OCPs

Samples undergoing acid purification and agitation for OCP analysis

My last week at the University of Liege has arrived, and I’m working hard to ensure that all the PHATS team’s labwork is complete before I leave to return home. All of our samples are now ready for analysis that will let us detect PCB and PBDE levels in our Scottish grey seals. PCBs and PBDEs are two types of the many persistent organic pollutants (POPs) that are present in our environment. I am currently working on preparing our samples for another type of analysis that will enable us to detect a third kind,  OCPs. As POPs in our environment, and PCBs in particular, are still currently in the news after the recent revelation of just how highly contaminated with PCBs some marine mammals are becoming, I thought I’d spend this blog introducing the three types of POP I work on and why they are so problematic.

Samples after acid purification, showing the clear fraction I need to collect for OCP analysis

PCBs, or polychlorinated biphenyls, are pollutants that are made up of two linked rings of carbon atoms with a varying number of hydrogen and chlorine atoms bound to the rings at different positions. There are many possible combinations of the number and locations of the hydrogen and chlorine atoms binding to the rings, and these give rise to the large variety of PCBs (called congeners) that exist. Approximately 130 different types of PCB are found in commercial products, and they can be divided into two groups (dioxin-like and non-dioxin-like) based on their structure and toxicity.  PCB production was banned in the USA in 1979 and by the Stockholm convention (signed by over 150 countries worldwide) in 2001, however they persist in our environment due to their slow degradation rates. One of the main reasons PCBs were previously manufactured and used in industry was their inert properties; only incineration at high temperatures can safely destroy them. Previous uses of PCBs include in coolants and lubricating oils, paints and electric wire coatings.

Orca have some of the highest measured POP concentrations in an organism worldwide due to their high position in the food chain.

PBDEs, or Polybrominated diphenyl ethers, are also made up of two carbon rings, but they have bromine bound to the rings rather than chlorine. The fewer the bromine atoms per molecule of PBDE, the more dangerous they are considered to be as congeners with between 1-5 bromine atoms bioaccumulate more effectively in living organisms. PBDEs are still being manufactured and widely used in many man-made products, the Stockholme convention which banned PCBs only restricted the production of some PBDEs. Some states in the USA have begun prohibiting their manufacture and use in the last decade however. PBDEs are flame retardant and are therefore commonly incorporated into electronics, plastics, fabrics and other building materials.

Bald eagles severely declined in the mid 20th centuary until the ban on DDT use in the USA. Bioaccumulation of the pesticide up the food chain affected the formation of their eggs, leading to thin shells that broke under the weight of an adult incubating them.

OCPs, or organochlorine pesticides, contain carbon, hydrogen and at least one bound chlorine atom but do not contain carbon ring structures like PCBs and PBDEs. There are many different types of OCP, however arguably the most well known is DDT (Dichlorodiphenyltrichloroethane) which was heavily used as a pesticide across the world to kill insects for both agricultural and disease control purposes. The famous book ‘Silent Spring’, written by Rachel Carson in the 1960s, is all about OCPs and the negative impact overuse of pesticides has on the environment. The production and use of some OCPs like DDT and heptachlor has been strictly limited by the Stockholme convention. Due to their efficiency at killing insects, their use is still permitted in some circumstances, such as the use of DDT to control mosquitoes that carry diseases like malaria.

POPs have been connected to a wide range of negative health impacts in both people and wildlife, and chronic exposure to any type of POP will cause problems for any organism. All POPs are carcinogenic (cancer causing) and are potent endocrine disruptors, interfering with growth and development, immune function and reproductive systems. There is growing evidence that POPs impact on obesity, leading them to be labelled as ‘obesogens’. The PHATS project I am part of is hoping to uncover some of the underlying physiological and genetic mechanisms that influence fat tissue function and determine how POPs can interfere with these processes. By studying a marine mammal species which has lots of fat and lots of bioaccumulated POPs, we can gain a better understanding of how these chemicals have such far reaching and devastating impacts on our health and the environment.

One of the PHATS team study animals from the Isle of May 2016, ‘Mike’, a newly weaned grey seal pup. Even though she is only a month old, ‘Mike’ will likely have high concentrations of POPs in her tissue due to the high position in the food chain (trophic level) seals occupy in the UK and the fact that mothers pass a large proportion of their accumulated pollutants onto their infants via their milk.

Liege 2017 – PCBs in the news, the most contaminated whale in the world

Purification columns being prepared with hexane for the lipid extracts from our blubber samples to be added.

It’s been another busy week here in chemistry labs at the University of Liege. I’ve completed extracting all the PHATS team’s blubber samples for persistent organic pollutant (POP) analysis, and now am moving on to the purification part of the sample preparation process. I’ve only got two weeks left to get all the sample preparation completed, so hopefully all the lab work will go according to plan! The purification process isn’t too complicated but it does have lots of time consuming steps, from multiple standard spikes, to acid clean-up on columns, to concentrating the samples down via nitrogen evaporation. So it’s just a case of getting your head down and getting on with it all, as the sooner it’s done the sooner we’ll have some interesting results to look through.

Purification columns, with samples from Echo to Kilo undergoing acid clean up. You can see how ‘dirty’ the samples are from the brown/black sludge that builds up in the columns!

The results of POP studies are frequently worrying as well as interesting. A good example of this happened last week, when the Scottish Marine Animal Stranding Scheme (SMASS) got some lab results back showing the PCB concentrations in one of the stranded whales they had examined last year, ‘Lulu’, one of Scotland’s few resident orca. She sadly had one of the highest ever recorded concentrations of PCBs in her body, and there are concerns that the other members of her pod will have similarly high levels. Another interesting (and sad) aspect of Lulu’s case is that she had never produced a calf, despite the fact she was about 20 years old and orca usually have their first calves at around 14 years of age. It is well known that POPs negatively impact on individual health, including fertility, therefore it is possible Lulu failed to reproduce due to her high pollutant burden. Even more concerning however, is what might have happened to Lulu’s high PCB concentrations if she had produced a calf.

Orca pod with young calves. The females will unwittingly pass large proportions of their pollutant burden to their infants, meanwhile males will steadily accumulate POPs all their lives.

Female marine mammals pass approximately 60% of certain types of the pollutants they have accumulated in their blubber to their first calf, some passing through the placenta but the majority transferring via the fat rich milk marine mammals produce. Therefore, if Lulu had produced a calf, it also could have had one of the highest PCB burdens ever recorded in a marine mammal. Male marine mammals typically have much higher POP concentrations than adult females due to this phenomenon, although even male orca in populations considered to be ‘highly contaminated’ (251.2mg/kg) have far lower concentrations than Lulu did (957mg/kg). This sex pattern in pollutant concentrations is present throughout all marine mammals, and after first reproduction an adult female’s POP concentration will gradually decrease with each infant she produces. This means that infant marine mammals are typically exposed to dangerously high concentrations of POPs as soon as they are born. Interestingly, in the two marine mammal species that appear to show menopause (orca and short-finned pilot whale), upon reproductive senescence a female’s POP concentrations begin to increase once again.

Humpback whale eating sand eels off the coast of North America. POP concentrations in fish eating marine mammals are usually lower than those that eat other marine mammal species to survive, but are also effected by how industrialised the environment where they forage is.

Another major cause of patterns in POP concentrations in marine mammals is their position in the food chain (their trophic level) and the region they obtain their food from. Orca represent a fascinating opportunity to study these patterns as through-out the species, there are different populations that specialise in eating either fish or other marine mammals, or in other words different orca populations can occupy different tropic levels of a food chain. Groups that eat marine mammals, such as seals, sea lions and porpoises, typically have over double the concentrations of POPs in them than fish eating groups. This happens because the pollutants have become concentrated up the food chain due to bioaccumulation, where a predator eating lots of smaller prey gets all the pollutants in each individual it eats. A whale eating lots of seals to survive will accumulate all the pollutants all the seals were exposed to, and all the pollutants all the fish those seals ate too. Meanwhile a fish eating individual will ‘only’ accumulate the pollutants from the fish it eats. Additionally, individuals that hunt in highly industrialised areas have higher concentrations than those in ‘pristine’ areas, because the more POPs that are in a local area, the higher the concentrations in all the organisms from the bottom of the food chain to the top.

Studying patterns of POP concentrations in different types of individuals can therefore lead to a better understanding of how these persistent pollutants ‘move’ through organisms and can be transferred into later generations. It is not hard to see why POPs continue to be a problem for animal and human health, despite being banned decades ago.

Liege 2017 – A brief introduction to blubber tissue

Minced blubber biopsies in cells ready to be capped and put through accelerated solvent extraction, to obtain all the lipids from a samples for further analysis.
Weighing a ASE vial after ASE is finished and the solvent has been evapourated off to calculate lipid mass in the sample. This is from one of Foxtrot’s biopsies from last year, and it had 0.42g of lipid in it, meaning 80% of the original biopsy was fat.

Well my first week at the University of Liege working with CART has flown by, and I’ve been working on the blubber biopsies we collected from the grey seals last year on the Isle of May. All the lipids (fats) need to be extracted from the blubber tissue before we can move forward with the pollutant analysis, so all the samples must be carefully prepared and put through Accelerated Solvent Extraction (ASE). This process uses high pressure and temperature conditions plus chemicals called solvents (like hexane and acetone) to remove all the lipids from the sample in the cells. This process gives us a completely liquid solution of lipids and solvents at the end of it, and we can then evaporate the solvent to leave just the lipids from our sample. This step is important as it gives the lipid mass of our sample, and allows us to work out how many nanograms of pollutant per gram of lipid in our sample there is (ng/g) . While ASE of our samples is an important step in the lab work, there isn’t really much more to say about it so I’m going to use the rest of the blog this week to give a brief introduction to blubber tissue, a crucial part of the anatomy of all marine mammal species worldwide.

Blubber tissue enables marine mammals to endure cold aquatic environments, like this Orca family living off the coast of Iceland.

All marine mammals, from the largest whale to the smallest seal, have a layer of fat underneath their skin called blubber. This layer of fat is extremely important for the survival of marine mammals for two reasons:

  1. It enables them to keep warm (thermoregulate) in freezing oceans.
  2. It provides a store of energy for individuals to utilise when they are not feeding, which happens in many marine mammal species at various points throughout their lives due to breeding or moulting.
A blubber biopsy from one of our seals. Blubber samples for pollutant analysis must be stored in glass or wrapped in foil to prevent loss of pollutants from the sample to any plastic they come into contact with.

Fat tissue deposits in all animal species perform these same two functions, however other species frequently have additional ways to thermoregulate (such as fur in land mammals) or do not endure long periods of fasting repeatedly while migrating or breeding as many marine mammals do. The importance of this tissue has lead to substantial blubber thickness evolving in marine mammals, and a stratified structure throughout the depth of the tissue is present so that it can perform both functions at the same time. Typically, blubber tissue can be roughly divided into three sections as you go from the part closest to the skin (the outer blubber) to the part closest to the inside of the seal (the inner blubber). The inner blubber is the most metabolically active, and this is where lipids are mobilised to provide energy for an individual when it either cannot find food or is purposefully fasting. The mid blubber is the most variable in thickness across individual marine mammals, and in thin individuals can be completely absent. It is thought it acts as a more long term storage tissue, and that its thickness is influenced by seasonal food availability. The outer blubber is typically of stable thickness within a species regardless of the nutritional state of an individual, and is thought to be primarily for thermoregulation. Hence even starving individuals will always have some blubber tissue to keep them from freezing, as the outer blubber is not mobilised as an energetic resource.

Blubber is a fascinating tissue to study and several different approaches can be used to analyse it in many contexts, like this recent study by one of my friends, Joanna Kershaw, who measured the hormone cortisol in blubber from porpoises to validate it’s use as a biomarker of body condition. The PHATS project I work on uses both established techniques (investigating pollutant concentrations) and novel protocols (the explant approach for tissue culture experiments that our team leader pioneered in seals) to make the most of the blubber samples we collect from our study animals to explore the prevalence of persistent organic pollutants in the marine environment and it’s impact on energy balance in seals.

A particulary tubby looking Charlie from last year’s study group on the Isle of May. Her fat reserves in her blubber layer will help her survive the tough first year at sea she faces when leaving the breeding colony.

MEANWHILE I am settling back into Liege life quite happily outside of the lab. I am not staying in the university accomodation this year, and have a lovely little flat not far from the campus to retreat to. In my time away from the lab I’m trying to keep up with the usual paper and grant writting that all resarchers need to keep on top of, plus greatly enjoying bebing reunited with the amazing macaroons they make here! Seriously, why can’t they be this good in Scotland…

Happiness is macaroons =D

PHATS project progress, ELISA validations and my return to Liege

Moulting grey seals hauled out on the Isle of May
Remember please send all ringed shag sightings to shags@ceh.ac.uk

Welcome back to my corner of the internet and the PHATS blog! The first four months of 2017 have flown by as the team has headed back into their labs to analyse all the samples we collected over the winter on the Isle of May grey seal breeding colony (to read about our fieldwork adventures, see these blogs here). I was lucky enough to escape to perform a survey of the Isle of May in January, to see if there were any grey seals that were moulting early in the year. There were plenty of them as it turned out, which bodes well for the fieldwork we are planning next year to try and look at moulting seal physiology. The island already looks so different to how it was when the seals breed there in the winter, much greener and all the seabirds are starting to come back. The cliffs were lined with guillemots, razorbills and shags; some were even getting started on gathering nesting material. The puffins had not returned yet, they arrive later in the year closer to summer, but we did see a few on the water during the boat crossing to the island.

Large grey seal haul out on the North East coast of the Isle of May
Is there anything better than well organised samples?

Inside the lab, I’ve been working on biochemical analysis of the cell culture media from all the blubber sample experiments last year (see here for more info) and am now two thirds through the samples we generated. By measuring the metabolic profiles of the various blubber culture experiments, we can see if the pollutant or hormone treatments had any impact on the blubber cells we collected from the seals. I’m also working on validating ELISAs (Enzyme-linked immunosorbent assays) to detect a variety of hormones in the blood samples we collected from the seals last year, so we can see if an individual’s hormone profiles are linked to their pollutant burden.

Using ELISAs on wild animal species like grey seals can be tricky, as they use antibodies as part of a binding process to detect the hormone you are interested in. These antibodies will have come from a specific species of mammal, usually rodent or domestic animal species that lots of scientists study, and the company making the ELISA kit will provide a list of species they know the kit works with. As a hormone’s protein structure is not always the same in different species of animals, the antibodies used in a widely available ELISA kit may not react properly with samples from unusual species that has never been tested with that kit before. Unfortunately seals often fall into the ‘unusual’ category, so we need to test the kits (validate them) before we use them to run lots of our samples, to make sure the results we are getting from the kits are accurate. There are several things to check when validating an ELISA, some of the most important are:

  1. Test for linearity. By diluting some of your samples (e.g. to half the concentration, then a quarter, then an eighth etc) you can make a serial dilution series to run on the kit. You can then see whether the curve the dilution series produces is parallel to the standard curve, which is what the kit uses to determine hormone concentrations. If the curve is not parallel to the standard curve, then the hormone in your samples is not binding correctly to the kit components.
  2. Test for recovery. By spiking a sample with a known quantity of the hormone you are interested in studying, you can tell how much the kit is detecting and how much is ‘lost’ during the analysis process.
  3. Test for consistency across kits and within kits. Many studies have lots of samples and need to use more than one kit to analyse them all. You must make sure the results of one kit are comparable to the others (inter-assay coefficient of variance), and the easiest way to do this is to run the same sample on each kit you use. You can then calculate the coefficient of variance across all the kits you have run. It’s also important to check the kit’s internal consistency (intra-assay coefficient of variance) by running one sample multiple times on a plate and seeing how similar the results are, and by running all samples in duplicate on the kit.

Our seal blood samples are proving to be rather tricky currently, and we’re still working on which are the best kits to use to measure the hormones we are interested in. The biochemistry analysis is going very well though, and we’re all looking forward to having some data to play with in the coming months.

Two ELISA plates at different stages of incubation. The different intensities of colour indicate different concentrations of hormone.

The biochemistry and ELISA work I’m doing is currently on hold however, as I have returned to the University of Liege in Belgium to work with the Centre de Recherche Analytique et Technologique (CART) to detect the amounts of persistent organic pollutants (POPs) in the blubber of our study seals from the Isle of May last year. We will be using Gas Chromatography – Mass Spectrometry (GC-MS) again, which requires a lengthy extraction and clean up process before the blubber tissue can be analysed (see these blogs from last year for more details) so I will be here for a month to work on our samples (and I will update the blog every week while I am visiting CART). It is always interesting, and more than a little sad, to find out how many pollutants all the seals have inside them after we got to know so well during the field season…

Unfortunately Alpha and Kilo, like all young marine mammals, will have large concentrations of POPs in their tissue from the high fat milk they drink from their mothers.

Au revoir to Liege and GC-MS basics

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The ion source of the mass spectrometer part of the GC-MS machine, with the old ion source on top waiting to be sent away for maintenance. The gas chromatograph part of the machine can be seen in the top of the photo, linked to the ion source by the part that has the ‘caution hot surface’ warning on it.

My time with CART at the University of Liege has now finished for this year! I completed all the lab work I needed to do which is great, but unfortunately the Gas Chromatography – Mass Spectrometry (GC-MS) machines are still down because of the heat. So we will have to wait to get all our pollutant results, fingers crossed things cool down in Belgium soon.

While they were trying to fix the machines during the last few days of my stay I got to see the insides of the GC-MS machines which was really interesting, they have so many intricate parts which you don’t normally get to see when they are running. The above picture is of a newly installed ion source in the mass spectrometer part of the machine (find out more technical details about ion sources here). On top of the machine is the old ion source in two bits that needs to be sent away for cleaning. In the picture below you can see the gas chromatograph part of the machine opened up to replace the column inside. The column is long and thin and looks like a coiled piece of wire (you can’t see it in this picture, its been removed but you can see where it joins to the inlet to the MS machine at the bottom right of the machine).

GC-MS machines work by coupling two detection methods together. The gas chromatography (GC) part separates out different substances in your sample by vaporising it (the gas phase) and then putting it through a column which is coated with substances (the solid phase) that interact with the vaporised sample. These interactions mean that different molecules in the sample elute, or reach the end of the column, at different times (the retention time). Substances can then be identified by how long they take to elute. However, some molecules have similar elution times under GC, called ‘co-elution’, which make telling them apart difficult from just this method. By combining the GC with mass spectrometry (MS) we can identify things with much greater accuracy.

MS works by ionising the vaporised sample eluting from the GC part of the machine by bombarding the various molecules with electrons (from the ion source, pictured above). The ions are then accelerated and deflected towards a detector, and measuring the mass-to-charge ratio of the ions enables us to identify which substances are present in a sample. Molecules that co-elute on the GC column will behave differently in the MS part of the machine, enabling us to tell them apart.

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The GC machine opened up and with the column removed for maintenance. The MS part of the machine is to the right, connected via the small, thin tube you can see in the bottom right of the GC chamber.

So I will bid ‘au revoir’ to Liege for the time being. I will hopefully return next year to analyse the new samples we will collect on the Isle of May this winter and from any seals we find moulting in the spring of 2017. When I get back to the lab I will be looking at the biochemistry of our study seals (measuring substances like glucose and free fatty acids in blood samples) and will also start preparing all the equipment we need for the big field season in October, like ordering a new CO2 incubation chamber that can be transported by boat to an island! The blog will be in hiatus until October when we’re about to set off for the Isle of May and exciting things are happening again. Expect weekly blog updates to return then…

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During the summer the Isle of May is open to visits from the public to see the huge bird colony there, but from October onwards the island is closed to protect the seals breeding there.

Labwork complete, finding food shops from Sart Tilman and heatwave

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Samples waiting for injection into the GC-MS machine. Each sample takes several hours to inject and run through the column.

With two working days to go before I’m due to fly home, I’ve finally completed all my laboratory work! I’m very happy and relieved to have got all our samples ready for analysis in the month I’ve had here, it means all the people here at CART have to do now is run the samples through the Gas Chromatography – Mass Spectrometry (GC-MS) machine and they can do that without me. As it turns out, the analysis will probably happen once I’ve left anyway as this week the summer has finally arrived here in Belgium and all the GC-MS machines are overheating because its so hot! So I will have to wait to actually get our results from all this work, the people at CART will just have to email the complete dataset to me once the analysis is finished.

Our samples will go through various GC-MS machines three separate times to detect different types of persistent organic pollutants (POPs),  once for polychlorinated biphenyls (PCBs), once for polybrominated diphenyl ethers (PBDEs) and once for pesticides. We already have one set of results, all the samples have been successfully run for pesticide concentrations. If the machines start working again I also may have some PCB results before I leave. In the last two days I have at the lab, I will hopefully get to discuss what the concentrations mean with my supervisor here, Dr Gauthier Eppe. Then I will head back home to St Andrews, ready to get back to work at the Sea Mammal Research Unit. That next field season is fast approaching after all…!

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Poor overheating GC-MS machine!

Meanwhile, back at the student halls… life has got a lot quieter now that the conference has finished, and the few people left are all trying to stay cool and avoid the thunderstorms that seem to roll round every few days. I’ve had to try and explain to a few people how to get to the nearest food shops to the halls over the last 3 weeks, so I’ve made a map of how to get to the two nearest supermarkets from a ‘google maps’ image. I’m sharing it here in case anyone else needs it! You take the 48 bus towards town from the main campus road outside the halls (take a right from the halls, then at the crossroads take a right again and the bus stop is just opposite RCAE- CRU building. Bus tickets are €2.10 for a single journey and its valid for several bus rides for 60 minutes). Then after a 10ish minute bus ride, get off at the stop opposite a Lukoil petrol station at a big intersection. Then follow the directions in the map below to get to either a medium sized supermarket (the ‘mini market’, which is acutally pretty big and has most things in it in terms of food supplies) or to get to a mall called Bell-Ile which has a big Carrfour inside. The Carrfour has everything you could ever need for supplies, including cutlery if you need to get kitchen equipment for using the cooking facilities in the halls. There are also loads of clothes shops and a pharmacy in the mall, and two good chocolate shops, Leonidas and Darcis. Belgium is of course famous for its chocolate so these two shops are great places to start sampling some! Darcis also sell the most amazing macarons, which I have become addicted to. The walk is only a 10ish minute walk from the bus station to Belle-Ile, so its a good idea to have a big bag you can sling over your shoulder to carry food shopping back in.

How to get to Bell Ile from Sart tilman
Directions to get to food shops from the 48 bus stop from the university halls

I went to the other local restuarant at the Sart Tilman roundabout this weekend as it’s now re-opened after the owners were on holiday. Marco Polo is a large, italian style place and the food there is really good! Its very affordable too, so I really recommend it to anyone at the halls. Its also the only place on the roundabout that is open at the weekends, and has quite late opening hours (open until at least 10.30pm, sometimes later depending what day it is) which is a nice change from everywhere else on the roundabout. It was really busy when I went so service was a little slow but everyone was really nice and made an effort to understand my terrible french and talk in a little english to help me order.

With only a few days left of my visit its almost time to start packing and trying to work out just how much chocolate I can fit in my luggage to bring home. Can’t believe how quickly time has gone, but it will be good to be back in Scotland!

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Ahh macarons, I think I will miss you the most…

GC-MS success, POPs in the marine environment and Sart Tilman life

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Fat extracts from our seal blubber samples after purification being concentrated under nitrogen gas so they can be injected into the GC-MS machine

To my great relief, the Gas Chromatography – Mass Spectrometry (GC-MS) analysis we ran at the end of last week worked and we detected PCBs and pesticides in all of the samples that we ran. This means that I could throw myself completely into churning through all 55 of my remaining samples, to try and get them extracted, purified and analysed before I am due to go home in just under 2 weeks time. I am making good progress so far and everything seems to be on track to meet this deadline, fingers crossed this week’s lab work goes as well as last weeks.

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‘Cells’ containing my blubber samples ready to go through accelerated solvent extraction (ASE) to get the lipids out of the blubber tissue ready for analysis. ASE uses high temperatures or pressure to recover the analyte of interest, and takes far less time than manual methodologies. Different solvent combinations can be used for different sample matrices or to extract different components.

Agreeing with previous studies on pollutants in high trophic level organisms like marine mammals, our study seals contained high concentrations of persistent organic pollutants (POPs). Many species of marine life have high levels of these chemicals in them, as despite the widespread ban on the industrial use of such substances, they continue to linger in the environment and all kinds of organisms, from low (like shellfish) to high (like tuna or dolphins) levels of the food chain. Perhaps most disturbing from our study is that our seals, at the time, had never been to sea and had never eaten any fish, yet they still have some of the highest POP concentrations CART (the laboratory I’m working with) have detected in a living creature. This is because, unfortunately, female seals must convert the fat in their blubber into fat rich milk to feed their pups, and the POPs they have accumulated in their blubber while they were at sea feeding gets transferred into their milk, which the pups then consume. POPs are a problem which are not going to disappear any time soon and studying the species which have the highest pollutant burden helps us understand how organisms are exposed to these chemicals and what the fitness consequences can be for individuals or exposed populations.

On a lighter note, life in the University of Liege student halls has got a lot more lively as its currently being used to house a lot of people who are here for, I think a conference but I’m not entirely sure. In preparation for this, the hall kitchens were all cleaned and are actually usable which is a massive plus, although walking up two flights of stairs with all my food/utensils is still less than ideal. I had a ‘room mate’ for all of 10 seconds too, well not a room mate but a person in the adjoining room who would have shared the shower with me (the shower with no door or way to close it off from the other person). She was obviously not thrilled with this idea or the room as she took one look at it and I haven’t seen her since, which is mostly funny but a little sad. I hope she managed to find somewhere else to stay that was better. It is hard to know what to expect before you arrive here, the university website is pretty vague about many aspects of the accommodation but they do have pictures of the rooms and state that showers are shared between two people (although they leave out the part where you better be ok with seeing each other naked all the time because there is no door). To be fair while things are extremely basic, everything does seem to work here, from the internet (although there is no wifi in the rooms, be sure to bring an internet cable) to the shower and if you do have a problem the 24 hour porters are extremely helpful, as long as you can get them to understand your God-awful attempts at french (some do speak some english and they all seem to be fluent in gesturing thankfully!).

I was listening to an obviously embarrassed student bringing an academic visitor to stay here this afternoon and heard them go through almost exactly the same thought process concerning food arrangements as I went through when I arrived, its good to know I wasn’t just being incredibly dense and/or demanding while trying to figure out the best way to feed myself. Their conversation could be condensed into the following:

  1. You could eat out, but all but one of the places that are walking distance to the university halls are closed on a weekend, and the one exception is also closed right now because it’s the summer holiday. So you should get a bus into town if you want to eat out.
  2. You could also cook your own food, because this place in theory has kitchen facilities, but actually what this means is it has hobs and sinks. That’s it. So as well as buying food from somewhere, you’ll need to buy utensils too. No, there is not a food shop within walking distance, you’ll have to catch a bus and find one in town somewhere. Oh and normally you can rent a fridge for your room but not right now, again because it’s the holidays and the students run the ‘rent a fridge’ service so they are only here on a wednesday night, for one hour.
  3. So what your saying is, I should stay in town, not here?

Having been here for 2 weeks now, I’ve figured out there are actually quite a few useful shops within walking distance but the opening hours seem very erratic and I’ve not been able to figure them all out yet (those listed online are not accurate!). Here are the places I’ve found useful in the last few weeks, they are almost all at the Sart Tilman roundabout just before you come into the university campus from town (if you are walking from the student halls, walk to the main road that goes through the university campus, go right and past the bus stop, take another right at the T junction and follow that road until you hit the small shops, cafes and the roundabout).

  1. The petrol station shop (technically called ‘station basri ozlu’). The people that run this petrol station clearly have the students in mind as their shop is full of essentials, no fruit or veg but there is fresh bread, a chiller with dairy and cold meats, canned things, snacks and a whole bunch of drinks from soft to alcoholic beverages. Good for when you’ve forgotten or run out of things and don’t want to have to catch the bus into town to get one loaf of bread. I can’t find out the opening hours but it’s never been shut yet when I’ve gone to buy things there!
  2. Le Campus. A nice little restaurant which serves tasty food at reasonable prices. The people are lovely and they took great care of me despite (or perhaps because of) the language barrier. Between the waitress, myself and my trusty french phrasebook I managed to order what I wanted and it was really good (yes the chips here in Belgium are amazing). Not open at the weekend, but open 10am – 8pm weekdays (I think?)
  3. The pharmacy. This is tucked away from the roundabout, if you’re looking at the church then there is a road to the left of it from the roundabout, take it and then take the next left to get to the pharmacy. Food shops in Belgium don’t sell anything remotely medical (no non-prescription drugs like paracetamol etc) so if you need anything like that, you need a pharmacy. Not english speaking when I went in, but very good natured and perfectly capable of understanding gestures (perhaps ‘plasters’ was an easy one though…).
  4. There is a good little flower shop on the roundabout to the right of the ‘tartines et chocolat’ bakery, perfect for buying bunches of flowers to take to give to people who have invited you round for dinner, which is a thing here in Belgium. The lovely lady who served me spoke good english and you can tell her how much you have to spend and she’ll just make something up for you.

Things I’ve not got to try yet on the roundabout include the bakery (tartines et chocolat) because every time I have tried to go there it has been closed and the ‘Marco Polo’ pizza restaurant, because its closed while the owners are on holiday. They are back next week though so I should get a visit in before I leave. There is also a burger/fries place (‘La Baguette Magique’) and a sandwich shop on the left as you walk to the roundabout which I’ve not tried, the fries place seems closed all the time too and the sandwich shop, while regularly open until 6pm, I’ve not needed.

So there are some options around if you know they are there! To finish, here is a picture of my room for reference. Only the bed, sheets, desk, chair, blackboard, bin and shelves come with the room, everything else you have to bring, buy or borrow.

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My home away from home at the university halls

Extraction, purification, concentration… and finally detection!

Solvent extracted blubber samples ready for purification from our scottish seals
Solvent extracted blubber samples ready for purification from our scottish seals

After a week’s worth of training and working to prepare a batch of our seal blubber samples, we’re finally going to get our first results tomorrow! It’s all very exciting, here’s hoping that everything has worked and we can actually detect some pollutants in them.

There is an incredible amount for me to learn here at the Center for Analytical Research and Technology (CART) at the University of Liege, but everyone at the lab has been fantastic. So many people are helping guide me through the numerous steps of sample preparation to clean up our filthy seal samples and concentrate them ready for analysis in the fancy Gas Chromatography – Mass Spectrometry (GC-MS) machine.

Essentially the protocol boils down to this. First you must extract the fat from the blubber sample (done using accelerated solvent extraction). Second you must evaporate off the solvent so you can find out how much fat was actually in your blubber sample. Third, you need to purify, or ‘clean up’ your sample so that as many of the unwanted substances are removed as possible before analysis (very important for dirty seal samples). Finally, you concentrate your sample into a small volume so it can be easily injected into the sensitive GC-MS machine. This description leaves out so many details of course, there are numerous washes, spiking with standards, evaporating and weighing steps to the protocols that I’m skipping over here for simplicities sake here. Needless to say after all the prep time I’m very keen to actually get some results tomorrow.

Purifying extracted fats from our blubber samples. It's not hard to see why the seal samples need cleaning up when you compare them to the quality control 'blank' sample that is on the left!
Purifying extracted fats from our blubber samples. It’s not hard to see why the seal samples need cleaning up when you compare them to the quality control ‘blank’ sample that is on the left!

Outside of the lab I’ve had a change of scenery. I have had to move into the University of Liege’s student halls for the rest of my stay, the lovely flat I was in last week is fully booked for the rest of July so I had to move on from there! It’s such a shame but oh well I’ll live. The halls are a big change, and to be honest they aren’t that well equipped or maintained; especially now it’s the summer holidays and most people have gone home. For example, there is nowhere to buy food from aside from a few vending machines outside of term times; the small canteen is closed for the summer. There are kitchens on every floor with hobs so you can cook things, but at the moment the majority are filled with dirty pans left by the recently departed students and aside from hobs, sinks and the occasional microwave that is it for kitchen equipment. That’s right, not even a fridge! Clearly the halls realised this was a problem at some point because they do run a ‘rent a fridge’ service, but again, it only really seems to run in term time. Thank goodness the team leader I’m working for at the university has a spare fridge he’s happy to lend me during my stay!

On the plus side the halls are less than 5 minutes walk from the university labs and the surrounding woods are very pretty to wander around in. I’m hoping to see some wild boar before I leave but they are pretty elusive when they want to be so I may not manage to find any.

I have also explored the city centre of Liege a little with the help of the lovely lab guys I work with, who kindly showed me around one evening and introduced me to as many Liege specialities as possible in one night. I had a great time eating Liege waffles, which are different to ‘Brussels’ waffles that come with tons of toppings so you need to sit down and eat one, Liege waffles tend to be coated or stuffed with chocolate or cinnamon so you can eat them on the go. We also visited ‘la Maison du Peket’ and I was blown away by the tasty Peket drinks they serve, Peket being a local type of fruit gin that comes in all sorts of incredible flavours and colours. Definitely try some if you are here and get the chance! For dinner we had ‘Boulets liégeois’, super tasty meatballs in a fantastic local sauce made from another regional speciality, ‘Sirop de Liège’ (an apple/pear/date syrup which I will try on my toast tomorrow). Of course this dish also came with the famous Belgian fries, so it was quite a spectacular supper. Finally we ended up in an ace quirky bar called ‘Le Pot au Lait’, another place to try if you are here for a night as the intricate, crazy decorations everywhere and the huge selection of drinks are not to be missed. I didn’t think to take any quick snaps while I was in the city enjoying myself, so I don’t have any to share which is a shame, no description would do all the extravagant artwork and sculptures in Le Pot au Lait justice.

It was good to get out for an evening though and see the city, especially as my workload is going to seriously ramp up next week as I tackle the bulk of my samples in the lab. Here’s hoping the analysis goes well tomorrow and I can get seriously stuck into prepping samples on Monday! Cannot believe I’m halfway through my visit already…

To Liege… and an introduction to the PHATS project

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Keeping an eye on the moulting grey seals resting on the Isle of May, Scotland

As June comes to a close the time of year for grey seals to moult in the UK draws to an end, so our team has stopped fieldwork and returned to the lab to focus on the analytical sides of the project. It won’t be long before the autumn breeding season rolls around though, so we have to make the most of these months while we can! With the end of the moulter work I have travelled to Belgium to work with the University of Liege on behalf of the PHATS project, and the lovely people at the Center for Analytical Research and Technology (CART) have given me a warm welcome so I’m getting along great even though my French is terrible!

I have come to CART to do a specific type of analysis that we can’t do back at home at the University of St Andrews or Abertay University. I am going to detect the amount of Persistent Organic Pollutants (POPs) that was in the seals we studied during the 2015 breeding season and the recent moulting season. This type of analysis requires a lot of specialised, complicated machinery so CART have offered their expertise and facilities to collaborate with us on the project. I have personally never run the types of extractions, purifications and detection methods that I will need to use to do this work, so I will have a lot to learn over the coming weeks.

I have not had much of a chance to explore Liege yet as I’m busy training at the lab, but so far I’ve had a peaceful time of it living on the outskirts of the city. Getting from St Andrews to Liege was relatively easy, the buses in Scotland (take the X59 and change at Ferrytoll to the 747 if you want to get from St Andrews to Edinburgh Airport) were on time and getting from Charleroi airport (sometimes called ‘Brussels South’ or something along those lines) was straight forward, the airport’s website has accurate and simple instructions for how to get to the nearby ‘Charleroi-Sud’ train station. I needed the ‘Liege-Guillemins’ station for my travel arrangements but there is another stop in the center of town called ‘Liege-Palais’ if you needed to go there. The buses here in Liege seem really good, if you have the street address of your start point and destination (try dropping points onto google maps of the areas you are in and where you want to go) this website is very useful even though its all in French, I can still navigate it anyway so I’m sure anyone can! The 48 bus is currently my best friend, it runs every ten minutes or so from the center of Liege (‘Liege Opéra’) to the University campus where CART is based, in Sart Tilman (any of the stops from ‘Amphithéâtres’ onwards will have you in the various parts of the campus). I’m staying in a lovely place through airbnb at the moment, apparently most students and academic visitors rent flats or ‘kots’ while they are here, but I will move into the student accommodation at the University soon due to limited availability. I’ve hear ‘interesting’ things about what it’s like there but we’ll hope for the best and prepare for the worst!

While I wait for some interesting developments to write about (I don’t think I’m going to touch the elephant in the room that is travelling to live in a European country four days after the UK voted to leave the EU, I’m still too sad/angry/in shock to write anything remotely sensible about it…) I’ll put a layman’s introduction to the PHATS project I’m working on here. If you’re already familiar with marine mammal pollutant science then feel free to skip it! Have a cute seal picture form the Isle of May on the house for reading this far though…

Grey seal pup
Grey seal pup

The PHATS project (Pollutants, Hormones & Adipose Tissue Science) is all about investigating how POPs impact the seals that live on our shores, focusing on how exposure to these chemicals may be affecting the seal’s ability to use their blubber stores properly. Blubber is a crucial part of any marine mammal’s anatomy, from the largest whale to the smallest seal pup. It not only keeps individuals warm in the ocean, but acts as an energy reserve so that when seals, whales or dolphins have to go without food (fasting), they don’t starve. Grey seals in the UK fast during two times of the year, when they breed in the late autumn/early winter and when they moult in the late spring/early summer.

POPs have been identified as obesogens, which are chemicals that prevent the fat tissue from responding to your body’s signals. Say you want to lose some weight, so you go on a diet and start doing more exercise. Your body should tell your fat tissue to start releasing the energy stored inside it to make up for the lower number of calories your eating. Your body uses hormones to tell the fat tissue it’s time to use up some of your fat for energy, but obesogens interfere with the way hormones talk to your fat tissue. So even though you are on a diet and doing exercise, obesogens could mean that your fat tissue can’t hear what your body is telling it to do, and you can’t utilise your fat tissue and energy reserves properly.

While other people are studying how much of an effect this may be having on human health, our study is looking at seals because marine mammals are know to have high concentrations of POPs in their fat tissue. This is because they are at the top of the food chain, and they bio-accumulate POPs because they eat lots of other animals that each contain a small amount of the chemicals inside them. POPs are especially dangerous as creatures can’t break them down or get rid of them quickly, so they persist for many years (hence the ‘POP’ name). In the past, scientists have linked POPs in marine mammals to problems with their immune systems and not being able to reproduce successfully, and we are one of many teams continuing to study how these pollutants are causing problems for marine mammal health.