WOBEC SOP Net sampling

Weddell Sea Observatory of Biodiversity and Ecosystem Change – WOBEC Standard Operating Procedures Technical Documentation December 2025 To be cited as Schaafsma, Fokje, Nils Van den Steen, Hauke Flores, and Anton Van de Putte. ‘WOBEC SOP Net Sampling’. Zenodo, 15 December 2025. https://doi.org/10.5281/zenodo.17936725. 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 April 2025 First draft H. Flores/F. Schaafsma 12/12/2025 Formatting N. Van den Steen Table of Contents 1 Description of net processing SOP ................................................................................................ 4 2 Sample sorting-collection-preservation procedure ............................................................... 4 2.1 Further sample processing: .............................................................................................................. 6 2.2 Processing of fish: ................................................................................................................................. 7 3 Length frequency analysis ............................................................................................................... 8 3.1 krill length-frequency analysis ........................................................................................................ 8 .................................................................................................................................................................................. 8 3.2 Measurements of salps ....................................................................................................................... 8 4 References .............................................................................................................................................. 9 5 Annex ........................................................................................................................................................ 9 Method responsible: Hauke Flores/Fokje Schaafsma 1 Description of net processing SOP This SOP describes the general process of sorting and preserving the catches of larger mesoand macrozooplankton sampling gear such as the Rectangular Midwater Trawl (RMT) 8+1 and de Surface and Under Ice Trawl (SUIT). The catches are used to determine the abundance and biomass of species in the mesoand macrozooplankton community, and the length-frequency distribution of krill and salp species. If desirable, samples of specific species may be taken from the catch for specific analyses, such as fatty acids/stable isotope analysis, gut content analysis, energy content analysis, DNA analysis, contaminant analysis etc. Such sample collection is described in detail in separate SOPs. If possible all macrozooplankton > 5 mm total length from RMT 8/SUIT shrimp net should be identified to the species level, prefferably from fresh material immediately after the catch, or later from preserved samples, using either a microscope or ZooScan. After sorting the larger organisms from the sample/subsample, the smaller constituents should be sorted using dissecting microscopes. Further splitting into subsamples might be necessary. If possible, the sample or subsample of other zooplankton should be preserved. If possible, length measurements should be carried out for the most abundant species. Mesozooplankton samples (RMT 1/ SUIT plankton net) will be analyzed entirely with the ZooScan . 2 Sample sorting-collection-preservation procedure 1. The mesozooplankton (e.g. RMT-1 and SUIT mesozooplankton net) sample(s) is concentrated with a suitable zooplankton sieve (< 300 µm mesh) and preserved on 4% formaldehyde in seawater. 2. The catch from each macrozooplankton net (i.e. RMT 8 from the single RMT or from the different depth strata of the MRMT, or the krillnet from the SUIT) is brought into a sorting laboratory and poured into a large plastic tray, labelled with the device operation and the net number clearly visible. 3. An overview picture is taken from each tray. 4. 5. In the RMT-8 / SUIT krill net sample(s): a) Large fish and jellyfish should be collected from the sample, photographed on neutral background with mm scale, and taxon, and no should be recorded on the catch datasheet. Fish should be put aside on ice in sorting tray until further processing. b) Jellyfish can be discarded after photographing, unless needed for dedicated sampling. c) The remaining catch should be concentrated on a sieve and either put into a sorting tray or, if the catch is large, split with a Folsom plankton splitter. One portion should be put in the sorting dish for further sampling, the other(s) should be stored in a Kautex jar filled with seawater and fixed with buffered formaldehyde (4% final solution)for taxonomy and quantification. . d) The catch in the sorting dish should be further processed. e) Spell out procedure from figure below. Splitter for 1-2 liter optioal for subsampling for different preservatives, picking etc. f) Volume with graded beakers, buckets, tubs g) at least 2, but if they differ by > 10% 3 subsamples h) counting preferably on board, subsamples preserved even if not counted. i) Take 3 subsamples, count at least 2, if they differ < 10%, that’s enough, but preserve all 3 subsamples. 2.1 Further sample processing: • Pick out desired species for specific analyses (CSIA, gut content, energy content, DNA analysis). Photograph each sample in a standardized way on a board with mm scale before preservation. Samples for specific purposes such as biomarkers, energy content etc. should be sorted and stored as described in the detailed processing SOPs. Record sample code, taxon, no, preservative, split factor (if applicable, i.e. 1.0, 0.5, 0.25, 0.125, 0.0625,.. ) and destination on the catch sheet. • Pick out and count Antarctic krill, other euphausiids and salps. Record sample code, species, no, preservative, split factor and destination on the catch sheet. If there is time, the fresh krill and salps should be measured and staged directly. After measurements, or when time is restricted, store the animals per species in a Kautex jar filled with seawater and fixed with buffered formaldehyde (4% final solution). Record identifier, taxon, no, preservative, split factor and destination on the catch sheet. • If large (> 100) number of gelatinous zooplankton such as chaetognaths are present, collect ~50 individuals. Photograph the sample in a standardized way on a board with mm scale before preservation. Record identifier, taxon, no, preservative, split factor and destination on the catch sheet. Store the sample in a Kautex jar filled with seawater and fixed with buffered formaldehyde (4% final solution). • If present, rare animals can be collected and stored in a Kautex jar filled with seawater and fixed with buffered formaline (4% final solution) for further taxonomic investigation. Photograph the sample in a standardized way on a board with mm scale before preservation. Record identifier, taxon, no, preservative, split factor and destination on the catch sheet. • Preserve all remaining macrozooplankton > 5 mm body size frozen (-20°C) in plastic bags with labels inside and outside, separately by taxon and net. Record sample code, taxon (remaining Zooplankton), no, preservative, split factor and destination on the catch sheet. • It is recommended to prepare a large plastic bag labelled with a separate bag ID, the device operation and the device, in which all frozen samples can be collected. • Make sure to label all samples taken. If there is no further processing time, the entire RMT-8 or SUIT shrimp net catch can be stored directly in a plastic bag and frozen at -20°C, after a photograph of the sample is taken in a standardized way on a board with mm scale. The bag should be labelled inside and outside. sample code, “Zooplankton” and the preservative, split factor and destination should be recorded on the catch sheet. 2.2 Processing of fish: Take a picture of whole fish on the measuring board. Measure total length and standard length. Take muscle and liver biopsy for CSIA, store in a pre-combusted pre-weighted Wheaton vial. Label & store at -80°C. Sample otoliths, if possible. Remove the stomach by cutting it between the oesophagus and the pylorus and store it in a Kautex jar filled with molecular - ethanol. Take two fin clips and put them in a eppendorf tube with molecular grade ethanol. Wrap the fish bodies in paluminium foil washed with Decon90 detergent inside plastic bag. Label & store at -20°C. Record sample number of the entire fish, each type of organ/body part sampled, no, preservative, split factor and destination on the catch sheet. Further details about fish processing are described in the fish processing SOP. Figure xx: Schematic catch sorting protocol for macrozooplankton from e.g. the RMT 8 nets or the SUIT shrimp net. If the catch is small, the sample will not be split and processing will occur as the second fraction in this scheme (right). In case catches are very large, the catch can be split more times before further processing. One fraction is stored in 4% formaldehyde, the other in the freeze (-20˚C). 3 Length frequency analysis 3.1 krill length-frequency analysis The standard length measurement is total length as defined by the Discovery method (AT) from the anterior margin of the eye to the tip of the telson without the terminal spines. The standard unit is given in mm below, with an accuracy of 1 mm size classes. Samples which contain less than 150 krill are used in total for length measurements and maturity stage identification. For large krill catches, a minimum of 150 krill shall be measured and staged. Krill sex and maturity stages should be identified using the classification of Makarov and Denys (1981, BIOMASS Handbook). Figure xx: measuring the length of Euphausia superba using the Discovery method (AT). 3.2 Measurements of salps If possible, all salps should be removed from samples smaller than 1 litre and counted. From larger samples, a random subsample of 1 litre should be taken by stirring the sample in a large bucket before removing the 1-L subsample. Please note the different species Salpa thompsoni and Ihlea racovitzai as well as the different forms (aggregate/solitary). If possible a minimum of 100 specimens per species should be measured. The internal body length from the mouth(branchial aperture) to atrial opening (atrial aperture) of a minimum of 100 specimens per species should be measured according to Foxton (1961) and CCAMLR(2000a). One should be noted there are two different forms: solitary and aggregate (figure xx). The internal body length should be measured to the mm below with an accuracy of 1 mm size classes. Figure 1. a) a solitary (asexual) salp, b) line drawing of a solitary salp indicating muscle bands and apertures c) line drawing of an aggregate (sexual) salp indicating muscle bands and apertures d) a chain of aggregate salps (Loeb & Santora 2012, SOKI Wiki, 2014c). 4 References Makarov, R. R., and Denys, C. J. (1981). Stages of sexual maturity of Euphausia superba Dana. Biomass Handb. 11, 2–13. 5 Annex 1. Catch processing protocol 2. Protocol measurements Euphausia superba 3. Protocol measurements salps 4. CCAMLR protocol