With our first field season behind us, it’s now time to start looking at our samples and data. Before our next field season, we hope to deduce the behavioural response of our 2018 whales to whale watching vessels. We also aim to successfully detect cortisol and other hormones in our blow samples. If we achieve these goals, we can take our research to the next step, safe in the knowledge that we can extract useful information from our fieldwork.

For these first couple of months, our main objective is to see what we can get out of our blow samples. With a short window and horrific weather this summer, we almost certainly do not have enough samples to correlate physiological stress with the presence of whale watching vessels. In fact, most of our blow samples belong to a single whale, Legolas, who often swam near to our land site and was a perfect study animal.

Iceland’s first blow sample

A beautiful blow sample from Legolas!

However, we can still use these samples to answer two key questions:

• Can we detect stress hormones in our samples?
• Can we compare hormone levels between different samples?

By teaming up with the Clinical Research Facility at the University of Edinburgh, we have a chance of answering ‘yes’ to both questions over the next few weeks and months. Get ready for some science…

How do you measure cortisol in whale blow?

Whale blow has three main components: seawater, condensed freshwater (from the air) and mucus. Since this mucus derives from blood plasma, it should contain many of the same molecules, including hormones- this has been shown in humans and many other animals, including whales and dolphins. Our focus is on cortisol, which correlates with stress in mammals.

However, this is hardly an easy molecule to find- it is quite small and found in tiny quantities in blow. It also has a similar structure to many other molecules. Therefore, we need to use a highly selective and sensitive analytical method, LC-MS

LC-MS stands for liquid chromatography- mass spectrometry. Essentially, it works in two steps

• Liquid chromatography separates organic molecules (including cortisol) based on physical properties (including size and how easily they dissolve in a specific liquid, called the solvent). Different compounds are separated at different times.
• As these compounds separate, they are passed into the mass spectrometer. Essentially, this charges molecules and and causes them to fragment. We can then detect different molecules based on their weight and fragmentation pattern.

Combining these two methods, we can very specifically measure cortisol. If cortisol is in our samples, it should be possible to detect it using LC-MS.

An LC-MS machine. Compounds are separated via liquid chromatography (left) and detected via mass spectrometry (right)

The challenge of sample dilution

So, we can hopefully measure the amount of cortisol in each sample. However, we have a major issue of dilution here. We don’t know how much seawater and how much condensed water has mixed with the mucus. Until we calculate this, we do not know the original cortisol concentration. Since this dilution will vary between samples, we therefore cannot compare our cortisol values between samples.

Studies in humans and other animals have also encountered this problem. This has led to the search for a normaliser- a molecule with known concentration in mucus. Whilst no gold standard has yet been found, candidates include urea (the waster product found in urine, which also circulates in blood) and salt. We will test these two normalisers for our blow samples.

Preparing a blow sample for analysis. The challenge lies in measuring sample dilution

In summary, we are optimistic about our chances of gaining useful information from blow samples. By refining our methods now, we can hopefully break new ground, using blow samples to show the response of whales to human presence.

If you have more specific questions about our work, please email info@whalewise.org. If you are interested in our work or passionate about whale conservation using new technologies, please visit our crowdfunding page: www.gofundme.com/whalewise. We would be grateful for any donations, no matter the size!