WOBEC SOP Under Ice Water Ruttner

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, Karley Campbell, Sebastien Moreau, Anette Wold, and Anton Van de Putte. ‘WOBEC SOP Under Ice Water Ruttner’. Zenodo, 15 December 2025. https://doi.org/10.5281/zenodo.17936920. 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 30 October 2024 SOPs for sampling of zooplankton M. Lenss K. Campbell S. Moreau A. Wold 12/12/2025 Formatting N. Van den Steen Table of Contents 1 Description of sampling gear .......................................................................................................... 3 2 Sampling gear deployment .............................................................................................................. 3 2.1 Deployment ............................................................................................................................................. 3 2.2 Post-Deployment................................................................................................................................... 4 2.3 Common Issues ...................................................................................................................................... 4 3 Sample sorting-collection-preservation procedure ............................................................... 4 3.1 Chlorophyll a ........................................................................................................................................... 4 3.2 Particulate organic carbon / nitrogen (POC/PON) .................................................................. 4 3.3 Flow cytometry ...................................................................................................................................... 5 3.4 Phytoplankton taxonomy................................................................................................................... 5 3.5 Inorganic nutrients ............................................................................................................................... 5 4 Sample analysis method ................................................................................................................... 6 4.1 Chlorophyll a ........................................................................................................................................... 6 4.2 Particulate Organic Carbon/Nitrogen ........................................................................................... 6 4.3 Flow cytometry ...................................................................................................................................... 6 4.4 Phytoplankton taxonomy................................................................................................................... 6 4.5 Inorganic nutrients ............................................................................................................................... 7 Method responsible: Sebastien Moreau 1 Description of sampling gear Under-ice water sampling is achieved using a hand-held Rutner water sampler with a rope and messenger through a large hole (≥ approx. 25 cm) in the ice. This hole should be made with an ice auger and can be specially made for use by the biology team (i.e., water and net sampling) or the hole used by the physics team for MSS, ROV, etc., deployment can be used. Alternatively, water can be sampled from the side of the ice floe if that is the best option. Processing of several parameters taken from under-ice water 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 proper chemical storage systems should be decided on and implemented before the cruise, including locked chemical storage cabinets and designated workspaces for radioisotopes. Chemical Parameter Safety consideration Glutaraldehyde Taxonomy and flow cytometry Carcinogenic Formaldehyde Taxonomy Carcinogenic and mutagenic Hydrochloric acid (HCl) Chloropyll a Corrosive Carbon-14 Primary production (P vs I curves) Radioactive 2 Sampling gear deployment 2.1 Deployment Prepare for station by ensure the Rutner sampler is clean (freshwater rinsed), the rope is labelled at the desired depths (see below), and that you have at least one back up messenger. Open the Rutner sampler, lower it to the desired depth, wait a moment (approx. 10 seconds), then carefully close the messenger around the rope and release it, making sure not to drop the messenger into the water. You should feel the Rutner sampler release and close. Haul the sampler back up to the surface and fill sampling bottles with sample, taking care to sample rinse all bottles 3 times before filling. Parameters taken from Rutner samples will be for Chlorophyll a POC/PON, flow cytometry, phytoplankton taxonomy, and inorganic nutrients. This requires roughly 4 L of water to collected at each sampling depth. Suggested sampling depths are 1 m, 5 m, 10 m, and 25 m. 2.2 Post-Deployment Take all samples back on board and to the lab for further processing as outlined below. Rinse the Rutner, rope, and messenger with freshwater and store with the other ice equipment for the next station. 2.3 Common Issues Remember to rinse all equipment used on the ice with freshwater after sampling. Be careful with messenger so as not to lose it in the ocean. 3 Sample sorting-collection-preservation procedure 3.1 Chlorophyll a • Filter up to 1 L of sample depending on biomass (a light color on the filter is enough) onto 25 mm GF/F filters. Remember to gently mix the bottle by inverting three times before filtering to ensure that particles are suspended. Also ensure that the mesh side of the filter is facing up on the filter holder, not the soft waves side (back side). • Filter under low vacuum pressure (about -30 kPa) and cover the tops of the funnels with aluminum foil when filtering. Using a squirt bottle, rinse the funnel by quickly swishing filtered seawater around the funnel three times once the sample has been filtered. Take care not to let the filters dry out. • Use forceps to lift the filters into plastic tubes for extraction. • Add 5 mL of methanol to the extraction tubes, close with lid, and cover with aluminum foil to block light. Place in refrigerator (dark, +4°C). • Allow pigments to extract into methanol for 12-24 hours, making sure to note the start and end time of extraction. • Rinse filtration funnels and manifold with freshwater between stations. 3.2 Particulate organic carbon / nitrogen (POC/PON) • Filter up to 2 L of sample (depending on particle concentration) onto precombusted 25 mm GF/F filters. Filters should be combusted at 450C for 12 hours before the cruise (Moran et al., 1999). Remember to gently mix the bottle before filtering to ensure that particles are suspended. Also ensure that the mesh side of the filter is facing up on the filter holder, not the soft waves side (back side). • Filter under low vacuum pressure (about -30 kPa) and cover the funnels with aluminum foil when filtering. Using a squirt bottle, rinse the funnel by quickly swishing filtered seawater around the funnel three times seawater once the sample has been filtered. Take care not to let the filters dry out. • After filtration, use forceps to carefully place GF/F into Pall filter slides. Place slides into oven set at 60°C and allow them to fully dry (about 24 hours). • Store samples at room temperature. Wrap together filter slides from one station in aluminum foil and keep them in a labelled Ziploc bag. • Prepare a reference filter (blank) for each station by filtering filtered seawater onto a pre-combusted filter. The amount of filtered seawater for a blank sample should be about the same as used when rinsing the funnels, so three quick swishes with the squirt bottle around the funnel. The reference filter will get a normal running number, so make sure to make a note on the CTD log sheet to avoid confusion with numbering on following casts. 3.3 Flow cytometry • Prepare for station by adding 38 µl of 25% glutaraldehyde into each pre-labeled 2 mL cryovials (3 per depth) under the fume hood. • Rinse a 50 mL falcon tube three times with sample filtered through a 40 µm cell strainer. Then fill the falcon tube with about 40 mL of filtered sample. • Pipette 1.8 mL of filtered sample into the prepared cryovials. • Fix the samples for 2 hours in the fridge and then snap freeze in -80°C. Store samples in cryobox at -80°C. 3.4 Phytoplankton taxonomy • Decant 190 mL sample into a pre-labelled brown glass (or plastic) bottle. • Bring samples back to the lab for fixation with aldehyde mixture under the fume hood. Add 0.8 mL of 25% glutaraldehyde and allow to fix for approx. 5 min. Thereafter add 10 mL of 20% hexamine-buffered formaldehyde. The final concentration is 0.1% glutaraldehyde and 1% formaldehyde in a 200 mL solution. • Store the samples in cool (+4C), dark conditions. Do not freeze. 3.5 Inorganic nutrients • Set up a clean working space and use vinyl gloves. Acid wash by soaking for 12-24 hrs in 10% HCl sampling vials(20 mL scintillation vials), syringes, and swinnex before sampling. Triple rinse sampling vials, syringes, and swinnex with MiliQ after acid bath and before use. • Assemble a swinnex with a pre-combusted GF/F filter. Take up a small amount of sample to rinse the syringe. Repeat three times. • Take up a full syringe of sample (about 60 mL), push out air, and attach swinnex. Using steady but light pressure, gently push sample through the filter at a drip-dripdrip speed. Rinse the filter with sample, and then rinse the sampling vial and cap with filtered sample 3 times. • Fill sampling vial with filtered sample, leaving a small amount of headspace for freezing. • Store samples at -20°C. 4 Sample analysis method 4.1 Chlorophyll a • Chl a samples can be processed on ship using a Turner Triology Fluorometer (Turner Designs, USA; https://www.turnerdesigns.com/trilogy-laboratoryfluorometer) 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. 4.2 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). 4.3 Flow cytometry • Samples for flow cytometry should be stored and shipped to UiT the Arctic University of Norway for analysis on a flow cytometer. 4.4 Phytoplankton 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 (Utermohl, 1958; Edler and Elbrachter, 2010) • 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). 4.5 Inorganic nutrients • Samples for inorganic nutrients should be stored and shipped to UiT the Arctic University of Norway for analysis on a QuAAtro Autoanalyzer (SEAL Analytical Ltd, Wrexham, United Kingdom).