Category Archives: Science

The Winkler method

Background

Oxygen represents about 21% of the air and it naturally dissolves into the ocean through gas exchanges between the water and the atmosphere. The concentration of dissolved oxygen in seawater is an important parameter used by physicist, chemist and biologist in oceanography. It enables to study the water masses, the redox potential of a water column or the biological processes that consume/release oxygen for example.

The Winkler method is a rapid and accurate way to measure dissolved oxygen in water samples. It owes its name to its inventor Lajos Winkler, a Hungarian chemist, who made it up in 1888 while he was still a doctorate student. The method was then improved in 1965 by Carpenter.

Filling 225 BOD right after the 5am sampling ...

Filling 225 BOD right after the 5am sampling …

Winkler equipment

Winkler equipment

Special materials

  • BOD Bottles: glass bottles specially designed to carry out the Winkler test. Our enemies are air bubbles and the special shape of these bottles makes easier the exclusion of these bubbles that will affect the accuracy of the oxygen measurements. All the bottles used have been numbered and calibrated so the exact volume of each BOD is known;
  • Reagent 1: MnCl2 (manganese (II) chloride solution);
  • Reagent 2: NaI, NaOH (sodium iodine basique solution);
  • Reagent 3: H2SO4 (sulphuric acid);
  • Sodium Thiosulfate: NaS2O3;
  • Metrohm titrator;
  • Computer & Tiamo software;
  • Teflon plunger

In theory

The objective is to estimate the production and the respiration of the planktonic community inside the mesocosms. As we study two oxygen-related biological processes, we are going to estimate them measuring oxygen differences after light or dark incubations.

In practice

The sampling of the mesocosms has to be done before the sunrise (5 am ready to leave the harbour)! Back on land, the BOD are filled with the seawater collected and a part of the bottles is directly fixed (means that the dissolved oxygen is trapped) adding reagents 1 & 2. The amount of oxygen measured is the initial concentration (T0), the initial state of the system. The rest of the BOD are incubated in the dark or in the light for 24H. After that time period, these bottles are also fixed and the concentration of oxygen is measured.

The oxygen difference between T0 and T24 light incubations represents the production while the difference between T0 and T24 dark incubations represents the respiration of the community.

Just a bit of chemistry…

From left to right: BOD filled with only seawater; the precipitate resulting from the addition of reagent 1 & 2; before the analysis, when reagent 3 has been added.

From left to right: BOD filled with only seawater; the precipitate resulting from the addition of reagent 1 & 2; before the analysis, when reagent 3 has been added.

The addition of reagents 1 & 2 in the sample (the fixation) traps the oxygen: in the basic environment created by the addition of NaOH, manganic hydroxides are formed resulting in a brown precipitate. The dissolved oxygen oxidize the manganese and convert the Mn(II) hydroxides into Mn(III) hydroxides, so the amount of Mn(III) actually corresponds to the amount of oxygen. This first reaction needs at least 5h to take place. After that, the sample is acidified just before the analysis. In an acid environment, the manganic hydroxides dissolve. The Mn(III) released oxidise the iodide ions (I-) previously added (reagent 2) to iodine (I2). Finally, the iodine is titrated by the thiosulfate (iodine is reduced and thiosulfate oxidised), the orange colour of the sample disappear slowly during the titration. At the end, the equivalent volume of thiosulfate is determined by the titrator and as we know that four moles of thiosulfate react with one mole of oxygen, the amount of oxygen in the sample can be determined. Considering the exact volume of the bottle (the BOD have all been previously calibrated) the oxygen concentration can be computed in µmol/L.

The advantage of getting up at 4:30 am to go sampling…

The advantage of getting up at 4:30 am to go sampling…

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All you need is…(2)

The song says ‘All you need is love‘ in mesocosms experiment we’ll say ‘All you need is GLOVES’!! When we are sampling some of the parameters need to be protected from us. That maybe sound strange but we are a source of contamination because of our skin and hairs. To measure some of the nutrients (ammonium, nitrate, nitrite, phosphate, …) as well as metals. So, the best way we could sample would be cover from head to toe and change every day… but not very doable as we have to wear sailing clothes!

To minimize the contamination, we have few things to do. First of all is to wear gloves but also avoid to touch our hair or skin (not very easy when we want to itch or to blow its nose!). The boxes have to be rinsed every day and even on the cubis, the back of the box with the sampler has to be rinsed into seawater before placing the sampler on it…. All a clean process taught by Cécile and Justine the two “clean room’s” scientists.

When Cécile and Justine aren’t dressed with nice sailing clothes they are with white labcoat, ‘charlotte’ to avoid contamination with hairs and overshoes…all this to avoid contamination. Justine, is measuring nano-nutrients which are the elements used by phytoplankton to growth but very easy to contaminate. She already explained her part during the previous experiment.

NB: pictures for this post are coming soon…

Trace elements at sea’s skin (by Alina)

Most of the people when studying ocean are sampling into the water column. However before entering into the oceans a skin have to be crossed. The few micrometers separating the water column and atmosphere is called microlayer and many interesting process occur. Alina explain us how to sample the microlayer and analyze trace metals (metals elements with very low concentration).

Sampling the Microlayer:

Microlayer sampling system during an experiment in 2011.

Microlayer sampling system during an experiment in 2011.

I have devised a new sampling method to get microlayer samples for trace metals.  The sampler is a quartz tube that is dipped vertically into the water; then slowly raised out of the water vertically. The water that drips off the tube is, in theory, the microlayer and is collected into a funnel that is connected to a receiving bottle.  Normally sampling the microlayer is done on calm days with little to no wind and waves.On Friday March 1 however, we had a treat and it was windy! It is good to get days where we think there is no microlayer (or it is well mixed) to compare to the calm days. The surface team had a fun roaster coaster ride on the cubi and earned the title of only team to sample that day!

Microlayer team sampling on the cubis during a very windy day (we had to cancel the other sampling)

Microlayer team sampling on the cubis during a very windy day (we had to cancel the other sampling)

After Collection Analysis:

The real fun begins for me after I leave this campaign…well, not really, but the bulk of sample analysis for me will be done in the labs on Florida State University’s campus and also at the National High Magnetic Field Lab, located down the street from campus.  So for those of you that are jealous of my afternoons off, don’t be because once I go home I will have 12 hour days for a few weeks.  Ever wonder how to analyze trace elements in nMolar concentration in seawater? Well, you have come to the right person! I have spent the better part (or worse depending on my mood that day) of the last 3 years learning techniques to determine the concentrations of trace elements in the dissolved and particulate phases of the water column.

Seawater extraction column

Seawater extraction column

The dissolved phase, for me, is what passes through a 0.4 μm pore sized filter.  I look at 10 elements, iron (Fe) being the most important as it is a major minor nutrient for marine critters.  The concentrations typically found in the dissolved phase are nMolar to pMolar in oligotrophic environments for Fe and the other elements I study (I have them all listed on my information page).  My research group uses an inductively-coupled plasma-mass spectrometer (ICP-MS) to determine the concentrations.  Now, anyone who has worked around an ICP-MS knows that sending straight seawater through it is a very bad idea. All the salts in seawater cause many issues and your data would not be reliable, if you manage to get any before your front end clogs up.  In order to get around the problem, my research group uses extraction columns which remove the metals from the seawater (Milne et al., 2010).  The column has resin inside that acts as a binding site for the metals.  The metals will adhere to the resin while the rest of the salts will pass through into waste when you get the pH of the sample right (something that takes a lot of practice).  Nitric acid (1.0 Molar) is then sent over the column that elutes the metals and sends them to a small collection vial.  The extraction process also concentrates the metals about 10 times, which makes it easier to analyze them on the ICP-MS.  This process (I left out a couple of steps for simplicity sake) takes 9 minutes per sample.  40 samples per day are about my limit as I must sit with the system the entire time.  The whole process is done in a Class-100 clean lab and I wear my little pastry chef outfit (hairnet, lab coat, gloves, clean lab shoes) to keep things as clean as possible.  The extracted samples are then ready to analyze on the ICP-MS.  Sounds like fun, right?

Me in my clean lab back in Florida

Me in my clean lab back in Florida

The particulate phase is a quicker and an easier process.  I save all my filters that my samples went through.  The filters are simply digested in order to get the total metal concentrations on the particles.  The digestion potion is a lovely combination of concentrated nitric, hydrochloric, and hydrofluoric acids (yum!) that dissolves the particle minerals.  They are then dried down, re-suspended in 0.34 Molar (2%) HNO3 and analyzed on the ICP-MS.  So, if I don’t die from digesting ~200 filters, I will be able to share the metal particulate concentrations.

Lots to do when I get home and not a lot of time to do it as I’m working on writing my Master’s thesis for an October 2013 defense and a publication on my microlayer sampler!  Good thing we didn’t sample today or tomorrow, maybe I can be productive?

Videoprofiling in the mesocosms

This year, we take the opportunity to be in Villefranche to use an instrument that has been developed at the Laboratoire d’Océanographie: the Underwater Vision Profiler.

The Underwater Vision Profiler 5 (UVP5; http://hydroptic.com/uvp.html, Picheral et al 2010) is an underwater intelligent camera developed by the Laboratoire d’Oceanographie de Villefranche-sur-mer to quantify the vertical distribution of macroscopic particles and zooplankton from 100 µm up to few millimeters in size. The smaller size limit is fixed by the optical resolution of the camera, whereas the larger size limit is determined by the volume of water illuminated per image. The UVP5 is compact (30 kg in air) and can operates as a stand-alone instrument with an independent power supply. Images are recorded at a frequency up to 6 Hz and analyzed on board by a dedicated software. Finally, Zooprocess (http://www.zooscan.com, Gorsky et al. 2010) is used to extract the the largest zooplanktons from the images and sort them into groups.

UVP view from underwater ( Picture: D.Luquet (OOV))

UVP view from underwater (
Picture: D.Luquet (OOV))

References

Gorsky, G., Ohman, M.D., Picheral, M., Gasparini, S., Stemmann, L., Romagnan, J.B., Cawood, A., Pesant, S., Garcia-Comas, C., Prejger, F., 2010. Digital zooplankton image analysis using the ZooScan integrated system. J. Plankton Res. 32, 285-303.
Picheral, M., Guidi, L., Stemmann, L., Karl, D.M., Iddaoud, G., Gorsky, G., 2010. The Underwater Vision Profiler 5: An advanced instrument for high spatial resolution studies of particle size spectra and zooplankton. Limnol. Oceanogr. Meth. 8, 462–473.

Return To The Mesocosms – Part One (by Lisa)

The Story so far:

In the Mediterranean Sea (not) far, far away………
It’s been 6 months since our intrepid scientists last visited the mesocosms. How will they cope with the change of location, the change in temperature & (more importantly) the change in sea temperature? There’s only one way to find out!!

Ok, joking aside, I’m sure that more than a few of us were a bit apprehensive about the change in sampling conditions; the last MedSeA mesocosm experiment took place in June/July last year in Corsica. The weather was mostly hot & sunny & most people sampled in shorts, tshirts & swimsuits with our feet dangling off the side of the cubis (our small floating sampling platforms). The water temperature was warm and comfortable and some even swam or kayaked to & fro.

Thus, a few of us greeted the news that these mesocosm experiments would be taking place in February with not a small amount of trepidation. What would be most practical to wear sampling? How could we still manipulate the integrated sampler & sampling bottles whilst still maintaining some feeling in our fingertips? Just how cold would pre-dawn sampling actually be?

Two days into the experiment, I can reveal that our fears were (mostly!) unfounded. After wrapping up warm & wearing either waterproof clothing or wetsuits it was as if we’d never been off the cubis.
Yes, it is cold, but it’s given us the chance to try various hot beverages from around Europe. So far we’ve had English Tea & Italian coffee, we look forward to Greek coffee tomorrow!

Now we can only hope that the air & water temperatures increase & bring on the start of the spring bloom – a period typically occurring in early spring where the abundance of phytoplankton increases significantly. The timing, size & length of a spring bloom can alter due to a variety of conditions such as nutrients & temperature, so it will be interesting to see the effect of ocean acidification. For me in particular it will be interesting to see the effects of the spring bloom under these conditions on various mechanisms within the nitrogen cycle – namely nitrogen fixation, nitrification & nitrate uptake.

In the meantime I’ll look forward to warming up with next hot beverage!