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(SOP) on Sampling and Analysis of Zooplankton Due Date of Deliverable Project Title: AQUACOSM: Network of Leading European AQUAtic MesoCOSM Facilities Connecting Mountains to Oceans from the Arctic to the Mediterranean Project number: 731065 Project Acronym: AQUACOSM Proposal full title: Network of Leading European AQUAtic MesoCOSM Facilities Connecting Mountains to Oceans from the Arctic to the Mediterranean Type: Research and innovation actions Work program topics H2020-INFRAIA-2016-2017: Integrating and opening research addressed: infrastructures of European interest Deliverable No 4.1.2: Standard Operating Protocol (SOP) on Sampling and Analysis of Zooplankton Due date of deliverable: Actual submission date: Version: V1.0 Main Authors: Deniz Başoğlu, Meryem Beklioğlu, Robert Ptacnik, Lisette de Senerpont Domis, Marko Reinikainen, Jens Nejstgaard, Gérard Lacroix This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 731065 Project ref. number 731065 AQUACOSM: NETWORK OF LEADING EUROPEAN AQUATIC Project title MESOCOSM FACILITIES CONNECTING MOUNTAINS TO OCEANS FROM THE ARCTIC TO THE MEDITERRANEAN Deliverable title Standard Operating Protocol (SOP) on Sampling and Analysis of Zooplankton Deliverable number D4.1.2 Deliverable version V1.0 Contractual date of delivery Actual date of delivery Document status Document version V1.0 Online access Yes Diffusion Public Nature of deliverable Report Work package WP4.1 Partner responsible METU, WCL, … Author(s) Deniz Başoğlu, Meryem Beklioğlu, Robert Ptacnik, Lisette de Senerpont Domis, Marko Reinikainen, Jens Nejstgaard, Gérard Lacroix Editor Deniz Başoğlu, Meryem Beklioğlu, Robert Ptacnik, Lisette de Senerpont Domis. Approved by Jens Nejstgaard (FvB-IGB) AQUACOSM – INFRA-01-2016-2017- N. 731065 2 EC Project Officer Agnès Robin Abstract This deliverable is a Standard Operating Protocol (SOP) that describes the methods for sampling and analysis of mesozooplankton from mesocosm experiments carried out in all aquatic environments (fresh and marine waters). It gathers best practice advice with a focus on sampling, counting and other analyses of mesozooplankton as well as Quality Assurance/Quality Control (QA/QC) practices. Use of this SOP will ensure consistency and compliance in collecting and processing mesozooplankton data from mesocosm experiments across the AQUACOSM community, in Europe and beyond. Keywords Zooplankton Analysis, Mesozooplankton, Sampling, Enumeration, Standard Operating Protocol, Freshwater, Brackish, Marine, Mesocosm AQUACOSM – INFRA-01-2016-2017- N. 731065 3 Table of Contents 1. Executive summary 6 2. Definitions and Terms 7 3. Cross References 8 4. Materials and Reagents 8 5. Health and Safety Indications 9 5.1 General Information 9 5.2 Safety Instructions for Sampling 10 5.3 Working and Personal Protection 10 5.4 Use, storage and disposal of reagents and chemicals 10 5.5 Use, storage and disposal of equipment 11 6. Environmental Indications 11 7. Sampling Zooplankton 12 7.1 Prior to sample collection 12 7.2 Required Strategies 12 7.3 Preparation and calibration of sampling equipment 12 7.3.1 Overview of equipment and instruments 12 7.3.2 Sample bottle preparation and preservation 12 7.4 Sampling Equipment 13 7.4.1 Plankton Nets 13 7.4.2 Sampling with plankton nets: 15 7.4.3 Using water samplers to collect zooplankton in mesocosms 16 7.4.4 Additional materials generally needed for zooplankton sampling 18 7.4.5 Sampling Containers and Sample Bottles 18 7.5 Sampling Design for Shallow and Deep Mesocosms 19 7.5.1 Sampling time and frequency 19 7.5.2 Shallow mesocosms 19 7.5.3 Deep mesocosms 20 7.5.4 Best Practice Advices on selecting the right sampling instrument 21 7.6 Best practice advice on preservation and storage 22 8. Quantitative Analysis of Zooplankton 23 8.1 Sample Preparation 23 8.1.1 Subsampling 23 8.2 Counting Procedure (Enumeration) 25 8.3 Best Practice Advice on subsampling and counting 26 AQUACOSM – INFRA-01-2016-2017- N. 731065 4 8.4 Taxonomy and nomenclature 27 9. Estimating the biomass 28 9.1 Crustacean Zooplankton 28 9.1.1 Measuring the length or dimensions of an organism 28 9.1.2 Length-weight relationships (Adopted from Bottrell et al. (1976) [48]) 28 9.1.3 Estimating the dry weight (Adopted from [49]) 29 9.2 Rotifers 29 9.2.1 Predicting dry weight using geometrical formulae (Adopted from [49]) 29 9.3 Estimating Mass as Carbon Content (Adopted from [49]) 30 9.4 Estimating biomass: other methods in literature 30 10. Size Distribution 31 10.1 Size Diversity 31 10.2 Normalised biomass-size spectrum (NSS) 32 11. Quality assurance and quality control 33 12. Appendix 34 Appendix A 34 Appendix B 36 Appendix C 36 Appendix D 38 Appendix E 40 13. References 41 14. Checklist for the next version 48 AQUACOSM – INFRA-01-2016-2017- N. 731065 5 1. Executive summary This Standard Operating Procedure (SOP) describes how to sample, identify and determine composition, abundance and biovolume of meso- and macrozooplankton from mesocosm experiments carried out in all aquatic environments (fresh, brackish and marine waters). It gathers best practice advice with a focus on sampling, counting and other analyses of mesozooplankton as well as Quality Assurance/ Quality Control (QA/QC) practices. It covers guidance on health, safety and environmental information, best practice advice on materials and methodology and QA/QC procedures to be followed during the sampling, analysis and counting of meso/macrozooplankton samples. It is designed to be compliant with the European Standard EN 15110:2006 [1]. Use of this SOP will ensure consistency and compliance in collecting and processing mesozooplankton data from mesocosm experiments across the AQUACOSM community, in Europe and beyond. Zooplankton are heterotrophic organisms living in open water whose distribution is primarily determined by water currents and mixing. The size of zooplankton ranges from a few microns to 20 µm (nanozooplankton mainly protozoans), 20-200 µm (microzooplankton, large protozoans and small metazoans), 200-2000 µm (mesozooplankton), and >2 mm (macrozooplankton) whereof those >20 mm are often distinguished as megazooplankton. This SOP focus on zooplankton >200 µm, as they are traditionally sampled with different methods than micro- and nanozooplankton and generally dominate the zooplankton biomass in mesocosm experiments. The dominant groups in mesocosm experiments in most water types are meso- and macrozooplankton such as crustaceas (dominated by copepods and cladocerans) often followed by rotifers [2]. In marine systems notable metazoan zooplankton normally also include Larvaceans and Chaetognaths. AQUACOSM – INFRA-01-2016-2017- N. 731065 6 2. Definitions and Terms Biomass the amount of living matter present in the mesozooplankton sample [3] Epilimnion or water above the pycnocline, i.e. thermocline (freshwater and marine systems) or halocline (marine) a in a stratified body of water [1] Upper mixed layer Fixation protection from disintegration of the morphological structure of organisms [1] Halocline vertical zone in the oceanic water column in which salinity changes rapidly with depth [4] Hypolimnion or water below the pycnocline, i.e. thermocline (limn./mar) or halocline (mar) a deeper layers in a stratified body of water [1] Littoral zone shallow marginal zone of a body of water within which light penetrates to the bottom; usually colonised by rooted vegetation [1] Pelagic zone body of water beyond the littoral zone [1] Plankton organisms drifting or suspended in water, consisting chiefly of minute plants or animals, but including larger forms having only weak powers of locomotion [1] Sampling site general area within a body of water from which samples are taken [1] (Sampling station) Stratified water freshwater (generally lakes/standing water) or marine waters with a strong density gradient (normally temperature and/or in marine systems salinity) resulting in an upper, normally warmer, mixed/isothermal layer floating on a denser, usually colder and or more saline, also isothermal water Subsampling collection of a sub-sample that consists of a known fraction of the total sample and that is representative of the quantity and species composition of the latter [1] Thermocline layer in a thermally stratified body of water in which the temperature gradient (metalimnion) is at a maximum [1] Zooplankton animals (heterotrophic organisms) present in plankton [1] AQUACOSM – INFRA-01-2016-2017- N. 731065 7 3. Cross References The SOPs that will be provided by AQUACOSM will be listed here in the following versions when the different SOPs are completed. The SOPs that will be provided by AQUACOSM will be for: 1. Phytoplankton (Deliverable 4.1.1) 2. Zooplankton (this SOP) 3. Periphyton (Phytobenthos) (Deliverable 4.1.3) 4. Water Chemistry (Physical and Chemical Elements of Water) (Deliverable 4.1.4) 5. High-Frequency Data Collection (Deliverable 4.1.5) 6. QA/QC (Deliverable 4.1.6) 7. Data Management (Deliverable 4.1.7) A general description for water sampling is covered under the Water Chemistry SOP. 4. Materials and Reagents Different preserving solutions with different areas of application are available in the literature. Some of the preserving reagents are summarized in Table 4-1. Table 4-1 The materials and reagents used in analysis of mesozooplankton Name and concentration Composition Storage Ethanol (normally 70-99 %) [1] Ethanol, water Solvent cabinet Instead of pure ethanol, cheaper methylated spirit can be used. If mesozooplankton is stored for molecular analysis, a solution from pure ethanol
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