WOBEC SOP Ice Coring

Weddell Sea Observatory of Biodiversity and Ecosystem Change – WOBEC Standard Operating Procedures Technical Documentation December 2025 To be cited as Lenss, Megan, Nils Van den Steen, Sebastien Moreau, Anette Wold, Karley Campbell, Maria van Leeuwe, Jacqueline Stefels, and Anton Van de Putte. ‘WOBEC SOP Ice Coring’. Zenodo, 15 December 2025. https://doi.org/10.5281/zenodo.17937014. Co-funded by the European Union (c) The authors This report is licensed under the Creative Commons Attribution International license Versions Date Comment Responsible Approved 4 December 2024 SOPs for sea-ice sampling of parameters related to protists and bgc M. Lenss S. Moreau A. Wold K. Campbell M.A. van Leeuwe J. Stefels 12/12/2025 Formatting N. Van den Steen Table of Contents 1 Description of sampling gear .......................................................................................................... 5 2 Sampling gear deployment .............................................................................................................. 6 2.1 Deployment: ............................................................................................................................................ 6 2.2 Calibration: .............................................................................................................................................. 7 2.3 Post-Deployment ................................................................................................................................... 7 2.4 Common Issues ...................................................................................................................................... 7 2.5 Workflow suggestion ........................................................................................................................... 8 3 Sample sorting-collection-preservation procedure: Protist sampling from sea ice10 3.1 Temperature ........................................................................................................................................ 10 3.2 Salinity .................................................................................................................................................... 10 3.3 Stable oxygen isotopes ..................................................................................................................... 10 3.4 Inorganic nutrients ............................................................................................................................ 11 3.5 Dissolved organic carbon (DOC) .................................................................................................. 11 3.6 Dissolved inorganic carbon / total alkalinity (DIC/TA) ...................................................... 12 3.7 Extracellular polymeric substances (EPS) ............................................................................... 12 3.8 Particulate organic carbon / nitrogen (POC/PON) ............................................................... 13 3.9 Chlorophyll a ........................................................................................................................................ 13 3.10 Biogenic silica ................................................................................................................................... 14 3.11 Ice algae taxonomy ......................................................................................................................... 14 3.12 Flow cytometry ................................................................................................................................ 15 3.13 Net community production using O2 optodes ..................................................................... 15 3.14 Ice algae culturing ........................................................................................................................... 16 3.15 Transmitted and incident irradiance measurements ....................................................... 17 3.16 Dimethylsulfoniopropionate (DMSP) ..................................................................................... 19 3.17 Net community production by recording uptake of the 13C stable isotope. ........... 19 4 Sample analysis method ................................................................................................................ 20 4.1 Temperature ........................................................................................................................................ 20 4.2 Salinity .................................................................................................................................................... 20 4.3 Stable oxygen isotopes ..................................................................................................................... 20 4.4 Inorganic nutrients ............................................................................................................................ 20 4.5 Dissolved organic carbon ................................................................................................................ 20 4.6 Dissolved inorganic carbon / total alkalinity .......................................................................... 21 4.7 Extracellular polymeric substances (EPS) ............................................................................... 21 4.8 Particulate organic carbon/nitrogen ......................................................................................... 21 4.9 Chlorophyll a ........................................................................................................................................ 21 4.10 Biogenic silica ................................................................................................................................... 22 4.11 Ice algal taxonomy .......................................................................................................................... 22 4.12 Flow cytometry ................................................................................................................................ 22 4.13 Net community production using O2 optodes ...................................................................... 22 4.14 Ice algal culturingii ......................................................................................................................... 23 4.15 DMSP .................................................................................................................................................... 23 4.16 Particulate organic carbon – 13C .............................................................................................. 23 Method responsible: Sebastien Moreau/Jacqueline Stefels 1 Description of sampling gear Biological sampling of sea-ice protist is done by collecting ice cores. Ice cores should be collected using a 0.09 m (inner diameter) core barrel (Kovacs Enterprise Mark II, USA) attached to either a hand corer or, ideally, a power drill. Researchers should work together in teams of 3-4 to collect and process ice cores. Responsibilities within the team should be clearly identified and all equipment and sample containers should be prepared and packed before arrival on station. It is essential that sampling containers are well labeled and organized before coring begins. Coring locations should be decided on upon arrival on site, with care taken not to walk over or disrupt the designated sampling location. Follow all safety protocols as prescribed by research vessel. Processing of several samples taken ice cores will include chemical use. A list of the necessary chemicals and their corresponding parameter is provided below. It is the responsibility of the individual using the chemical and processing the sample to operate in a manner that is safe for themselves, others on the ship, and the environment being sampled. Safety Data Sheets (SDS) should be readily available in the labs and and safety procedures as prescribed by the research vessel should be adhered to, including locked chemical storage cabinets and designated workspaces for radioisotopes. Chemical Parameter Safety consideration Saturated mercury chloride (HgCl2) DIC and CH4 Highly toxic Methanol Chlorophyll a Carcinogenic Glutaraldehyde Taxonomy and flow cytometry Carcinogenic Formaldehyde Taxonomy Carcinogenic and mutagenic Hydrochloric acid (HCl) Several Corrosive Ethanol Several Toxic NaOH DMSP corrosive 2 Sampling gear deployment 2.1 Deployment: • Ensure all equipment is ready, including core barrel, drill, shovel, saw, cutting board, sampling containers, notebook, gloves, measuring stick, ice thickness tape, ice auger, light measurement equipment, etc. • Choose the coring location. In the field notebook, take note of basic weather and time observations including cloud cover and time of day. • Prepare for the first ice core by taking 3 measurements of the snow depth 8 cm equidistant from each other (in a triangle with 8 cm sides) using a measuring stick. Note down snow measurements in the field notebook. • Remove snow over the coring location using a shovel. Then start drilling the ice core with the Kovacs corer and drill. Ideally, one person is responsible for coring and does not touch the core at all while another person has “clean hands” and wears vinyl gloves to extract the core from the barrel, measure, and cut it. • When ice core is retrieved, bring it to the cutting board. The cutting board should be set-up in a shaded location, for example a tent or in the shade of a cooler/box, and the ice core should be exposed to as little light as possible. The person in charge of cutting measures and notes down the core length before cutting the core. The cutting scheme should be decided on before the station, and a standard cutting scheme for biological ice cores is 0-5 cm, 5-10 cm, 10-20 cm and 20 cm sections and a standard cutting scheme for physical and chemical cores is every 10 cm. Zero on the ice core represents the icesnow interface. • Proceed in this way until all the way through the ice, using extensions for coring if necessary. • Take ice thickness and free board measurements at the core hole after extraction of the core using the ice thickness tape. 2.2 Calibration: Follow calibration procedures for the on-ice thermometer and salinometer that will be used in the lab following sampling. For the on-ice thermometer we recommend a Testo 720 temperature meter, see here for instrument information and a detailed manual. For a salinometer, we recommend a WTW ProfiLine Cond 3110, here is instrument information and a detailed manual. 2.3 Post-Deployment Once all on-ice measurements have been taken (ice cores, light, under-ice water, plankton nets), bring samples back to the ship for post-processing. Ice core sections for biological parameters should be pooled together and immediately diluted with filtered seawater at a 3:1 volumetric ratio for buffered melt. Buffered melt should take place in cool, dark conditions, e.g., in cooler jugs. For buffered melt, use seawater filtered through a 0.2 µm filter. The melted sea ice and filtered seawater solution can then be subsampled for all desired biological parameters, where measurements represent an average response of the bottomice community at that particular sampling location. Remember to freshwater rinse all equipment after sampling and MiliQ rinse melt containers between stations. Also remember to take final volume measurements of all ice core + FSW solutions before further sampling (i.e., filtration). 2.4 Common Issues Well organized and intuitive log sheets and labeling systems should be used commonly throughout the cruise. Ice core sampling plan including core division, roles, order of cores/sampling etc. should be discussed and decided on before station. The sea-ice biologists and physicists should discuss parameters interesting to both parties beforehand (i.e., temperature, salinity, microstructure, inorganic nutrients, etc.) to avoid double sampling. The number of cores taken per sample should be decided based on the volume needed for desired parameters/experiments. Keep in mind that a larger number of biological cores requires higher volumes of FSW. Preparing FSW for an ice station can be time consuming, set up a peristaltic pump and prepare FSW before sampling as much as possible. A suggested workflow is outlined below with a table including the number of ice cores needing to be sampled and corresponding parameters. Note that ice coring, water sampling, and net sampling can occur in parallel by small working groups (2-4 people each). 2.5 Workflow suggestion • Upon arrival at the ice station, note time, position, and weather conditions. Decide coring location. • Set-up for ice coring, delineate coring location and ask others NOT to walk over coring site. • Take light measurements using under-ice arm and LI-COR tripod. • Take temperature core, making temperature measurements using drill and thermometer. Work quickly to avoid warming of core. • Take all other ice cores, sectioning into appropriate sections along the way. • Take under-ice water samples. • Take net samples. Core name Number of cores Parameter(s) measured Post processing Temperature core 1 Temperature Processed immediately on the ice using thermometer Physics 1 1 Microstructure Bagged whole, taken on board and stored at - 20C Physics 2 1 Density Bagged whole, taken on board and stored at - 20C Chemistry core 1 Salinity, d18O, inorganic nutrients, DOC Cut into 10 cm sections and placed into prelabelled containers for unbuffered melt (i.e., no added FSW) DIC core 1 DIC Cut into 10 cm sections and placed in vacuum sealed bags for gas-tight melt Bio core 1 3 Chl a, POC/PON, EPS, and biogenic silica Cut following the biological sectioning scheme and bring onto ship for buffered melt (3:1 ratio with FSW) and post-processing (i.e., filtering and fixation) Bio core 2 1 Ice algae taxonomy, flow cytometry, and microscopy, DMSP Cut following the biological sectioning scheme, keep in the dark!, bring back into ship for buffered melt (3:1 ratio with FSW) and postprocessing Bio core 3 4 Primary production measurements (oxygen optode PI curve incubations) Cut using desired sectioning scheme, keep in the dark!, bring back into ship for buffered melt (3:1 ratio with FSW) and postprocessing Bio core 4 3 Ice algal culturing Scrape off the bottom 1 cm of the ice core where the algae is most concentrated and place into sample container. This will make a highly concentrated sample for culturing of ice algae. • Calibrate optode sensors immediately prior to each incubation using a 0% and 100% dissolved oxygen standards of 0.17 M sodium dithionite (Na2S2O4) and air saturated water (bubbled FSW for more than 10 minutes), respectively. This accounts for any sensor drift or photodegradation that may have occurred with extended use. The calibration solutions should be made fresh before each incubation. • Once calibrated and equilibrated, fit each bottle with an optode sensor, taking care not to pull the optodes up/down during place or incubation to avoid volume displacement and inaccurate measurements. • Incubate samples for 24-70 hours under continuous illumination and mixing. The 1 cm magnetic stir bars should be set to about 60 rpm. The oxygen concentration is recorded for each bottle at about 2 s time intervals throughout the entire incubation period. • To stop incubation, turn off optodes and remove from bottles. Measure average PAR using handheld PAR sensor at each bottle position by replacing optodes with PAR probe. • Pool all samples after incubation (except dark bottles) and filter for additional chlorophyll measurement. An additional nutrient sample can also be taken in order to determine net nutrient uptake (if done, a nutrient sample must also be taken at T0 from bulk sample). Rinse optodes with MIliQ and gently blot dry with Kim wipe to prevent salt damage and corrosion. Rinse incubation bottles three times with FSW after each incubation. 3.14 Ice algae culturing • Set-up LED strip lights in a section of a refrigerator (+4°C) by taping the LED onto the inner walls or shelving. Adjust the lighting to a very very low setting to force slow growth and prevent nutrient limitation. • Use a 0.2 µm sealed filter and a sterile 60 mL syringe. • Place syringe into F2 media bottle and draw up media. Hold the cap to the media bottle while you’re doing this step to avoid contamination of the cap. • Place filter onto syringe and filter media into a new and sterile culture flask (60 mL Falcon flask, for example). • Place re-filtered media into the fridge to cool. • Transfer ice scrapes from Bio Core 4 into the cooled culture flasks, taking care not to touch the inside of the flask. Seal with cap. • Place the culture flask back in the fridge as soon as possible after transfer. If possible, lay the flask horizontally so there is a larger surface area for the algae to grow on. 3.15 Transmitted and incident irradiance measurements • Program the LI-COR LI-1500 logger before arrival site visit/station for efficiency. The logger can be programmed with the provided sensor calibration, which is unique to each sensor used. Sensor calibrations are provided for measurement in air or water (i.e., under the ice), and each must be used separately for surface or transmitted measurements. • Calibration data is entered to the logger by selecting MENU > Sensors > Add New Sensor and selecting the model number LI-COR LI-193. A sensor out of calibration warning will appear if more than two years have passed since the date of the sensor’s last calibration. The sensor must be sent back to the LI-COR factory for recalibration. Double check that the sensor is satisfactorily calibrated before the cruise. • Calibration information can be found online by searching for the sensor’s serial number at CALIBRATION. All necessary calibration data must be entered for the sensor being used. • Detailed information about settings and configurations on the logger can be found online at LI-COR. • Mark out the area to be used for coring and (e.g., with flags/bamboo sticks). Take caution to NOT WALK over the sampling area. A pristine snow cover is required for representative light measurements. • Assemble the underwater arm and attach the LI-COR LI-193 spherical underwater quantum sensor. Make sure cable ends do not get wet and take caution not to damage the sensor. Turn on the logger and double check the configurations. • Briefly, the input selected on the logger should correspond to the input port where the LI-COR LI-192 sensor is attached. Take care to select air or water depending on which measurement is being taken, and that configuration is “Active” in the menu settings. Settings for these steps can be found by selecting MENU > Configurations > Add New Config. • Take snow measurements and then make a hole using the 10 inch auger. The hole should be made at the north side of the sampling area such that the deployed arm (which is 1.5 m out from the deployment hole) will be positioned facing south and will be in the center of the 3 x 3 m area. • Then insert the underwater arm in the auger hole with a LICOR sensor attached and pull on the string to position it upwards. Turn the sensor towards the sun and position it roughly 10 cm below the ice bottom by carefully bringing the sensor up until it lightly touches the ice, and then lower it 10 cm from that position. • Select the channel for measurements in water on the logger (should be preprogrammed before arrival on site). • Note down 3 to 5 measurements from the LICOR sensor. This represents integrated incident downwelling irradiance transmitting beneath the ice-water interface. Take note of variable weather conditions between measurements and these can have a big impact on reliable transmittance data. • Remember to freshwater rinse equipment after sampling. • Sea ice cores for any bio-optical calibrations (e.g. remote estimation of sea ice algal biomass) should be taken proximal-to the sensor location under the ice (i.e. in the center of the 3m x 3m sampling area) • Select the channel for measurements in air on the logger (should be preprogrammed before arrival on site). • To measure incident upwelling (albedo) and downwelling irradiance above the snow surface, attach the LI-COR LI-192 quantum sensor to a 1 m pole that is held level and approximately 1 m above the snow surface and facing the sun. A tripod set-up is optimal. Make sure to position yourself below the sensor to not create shadows. • Note down 3 to 5 measurements with the sensor facing upwards and 3 to 5 measurements more with the spectrometer facing downwards. This represents the incident downwelling irradiance and incident upwelling irradiance above the snow surface, respectively. • Also take note of weather conditions during measurements, which can have a big impact on reliable transmittance data. Take note of the cloud cover and how it is changing. • If sampling at the same sites consistently over a campaign, light measurements should be taken at a consistent time of day (e.g., always in the morning or always at noon or…). 3.16 Dimethylsulfoniopropionate (DMSP) • DMSP is a volatile and relatively insoluble trace gas and the concentration in the sample will be affected by prolonged contact with air. Therefore, sample with care. • Prelabel the sample bottles prior to sampling. • Take 10mL from each depth horizon and pipet directly in a 20 mL glass DMSP-vial. Add 50 µL of a 2.3µM D3-DMSP working solution for standard additions. • Add 1 pellet of NaOH and close firmly. Label appropriately and store upright in - 20°C 3.17 Net community production by recording uptake of the 13C stable isotope. • Start the experiment by adding 2 ml 13C-NaHCO3 from a stock solution (1 gram 13CHCO3 + 20 ml MQ) to ca. 2L of the melted sea ice. • Mix the tracer by gently shaking the bottle. This is important! • Take t0 samples - DIC: fill a 8ml amber bottle to the top. Store at 4˚C. - DMSP: use a 10ml pipet for 3 x 10ml in 20ml screw-top vials + 1 pellet NaOH. Store at -20˚C. - Chl.a: filter ca. 20ml; for further details see protocol • From the Nalgene bottle, transfer 250ml to 6 tissue culture flasks. • Arrange LED lamp adjacent to clear wall on chamber (see protocol for O2 optodes. • Arrange the tissue flasks and label consecutively from position one to position six, with the first position marking the bottle closest to the light (i.e., the clear chamber wall) in a pre-cooled and darkened chamber. The bottle at position six (farthest from the light) should be prepared with black electrical tape to prevent any light from entering in the bottle and will be used as the dark control. • Incubate samples for 8-10 hours under continuous illumination. Gentle shake the flasks every hour • To stop incubation, take the samples from the experiment in a fixed consecutive order for filtration. Make sure to note the time. Prepare and label alu foil before filtration. • Depending on the biomass 50-200ml is filtered over 2.5cm pre-combusted GFF Whatman filters using a low vacuum pressure (-30 kpa) under dim light conditions. Fold the filter 1x, pack in alu foil and flash freeze in LN2. • Store at -20°C • Complete the meta-sheet. 4 Sample analysis method 4.1 Temperature • Temperature measurements will be taken on the ice. No further analysis is needed. 4.2 Salinity • Salinity will be measured aboard R/V Polarstern using, for example, a pre-calibrated Profiline Cond 3110 portable conductivity meter (WTW, Germany). No further analysis is needed. 4.3 Stable oxygen isotopes • Samples for δ18O should be stored and shipped to Vrije Universiteit, Brussels, Belgium for analysis on a Perspective isotope ratio mass spectrometer (Nu Instrument, Ametek) coupled to a Gas Bench system. All samples should also be standardized against V-SMOW. 4.4 Inorganic nutrients • Samples for inorganic nutrients should be stored at -20C (see section 3.4) and shipped to UiT the Arctic University of Norway for analysis on a QuAAtro Autoanalyzer (SEAL Analytical Ltd, Wrexham, United Kingdom). 4.5 Dissolved organic carbon • Samples for DOC should be stored and shipped to the UiT the Arctic University of Norway for analysis on a Shimadzu ASI-V/TOC-V analyzer. 4.6 Dissolved inorganic carbon / total alkalinity • Samples for DIC/TA should be stored and shipped to the Institute of Marine Research in Tromsø, Norway for analysis using potentiometric titration with 0.1N hydrochloric acid on a Versatile Instrument for the Determination of Titration Alkalinity (VINDTA 3D, Marianda, Germany). 4.7 Extracellular polymeric substances (EPS) • Samples for EPS should be stored and shipped to UiT the Arctic University of Norway for analysis on a UV-Vis spectrophotometer and following the phenolextraction protocol of Dubois et al., 1956 (Dubois,M. and Gilles,K. A. and Hamilton,J. K. and Rebers,P. A. and Smith,F., 19561402659, Journal article, 28, Analytical Chemistry, (350–356), Colorimetric method for determination of sugars and related substances., (1956)). 4.8 Particulate organic carbon/nitrogen • Samples for POC/PON should be prepared for analysis by acidifying in fuming hydrochloric acid for ca. 12 hours before packing into tin capsules. • After fumigation and packing, samples will be sent to Vrije Univeristeit, Brussels, Belgium for measurement with an element analyzer (EA, Eurovector, Italy) coupled to an isotope ratio mass spectrometer (Horizon IRMS, Nu Instruments, United Kingdom). 4.9 Chlorophyll a • Chl a samples can be processed on ship using a Turner Triology Fluorometer (Turner Designs, USA) following the steps outlined below. • Turn fluorometer on at least 10 min and allow samples to warm to room temperature before taking first measurement. • Fill 10 mm cuvette with methanol and dry on the outside with a KimTech wipe, place in cuvette holder inside the fluorometer and take measurement. This is the pre-analysis blank measurement. • After taking first blank, transfer sample into a new 10 mm cuvette and dry on the outside with a KimTech wipe. Place in fluorometer and take measurement. Read the value and note down as Rb value (before acid addition). If sample is too concentrated dilute with methanol, making sure to note down dilution. • Take the cuvette out of the fluorometer and add 2-3 drops of 5% HCl, cover the cuvette with parafilm and mix gently by inverting 3 times. Wait 30 seconds to allow reaction to complete and take a new measurement. This is the Ra value (after acid addition). • Take measurements in this way for all samples, and then take a final blank measurement. • Dispose sample after readings into designated methanol waste container. • Fluorometer should be calibrated before or after the cruise with Chl a extract to derive calibration constants noted down for calculation of Chl a and phaeopigments on Excel. 4.10 Biogenic silica • Samples for the determination of the biogenic and lithogenic silica should be stored and shipped to the University of Brest, France for analysis following the NaOH/HF digestion method of de Master (1981). 4.11 Ice algal taxonomy • Samples for taxonomic analysis should be stored and shipped to the Institute of Oceanology, Poland using light microscopy. • Samples should be sedimented for 20-40 hours before analysis following the Utermohl method. • Taxa identification should be done based on morphological characteristics following e.g., Johansen and Fryxell (1985), Thomas (1997), Scott and Marchant (2005), and the online repository at the World Register of Marine Species (https://www.marinespecies.org/index.php). 4.12 Flow cytometry • Samples for flow cytometry should be stored and shipped at -80C to UiT for analysis on a flow cytometer. 4.13 Net community production using O2 optodes • Optode incubation data will be stored on the associated computer and should be backed up on at least one other location. No other processing is required. 4.14 Ice algal culturingii • Samples for ice algal culturing should be delivered to the MicrSiO laboratory at UiT the Arctic University of Norway for further growth and eventual experimentation. 4.15 DMSP • Pre-label glass storage bottles before starting sample preparation • DMSP: From each amber bottle, pipette very accurately (!) 10 mL into a 20 mL DMSP-vial with a 10 mL plastic pipet and a red pipetting balloon. You do not need to change pipettes for every sample (save the environment!!), but can use one pipet for all samples of one sampling day. In between each sample: rinse with milli-Q, followed by a few mL of the next sample. • Add 50 µL of a 2.3µM D3-DMSP working solution. • Add a pellet of NaOH and close firmly with a screw cap (wearing lab gloves improves the grip on the caps!). • store at -20°C. 4.16 Particulate organic carbon – 13C • Samples for production analysis will be sent to the University of Groningen for analyses with Cavity Ring-Down Spectroscopy analyser (CM-CRDS, with a G2101-I Analyzer, Picarro, California, USA).