SITES AquaNet 2017 Sampling and laboratory protocols

Transcription

1 SITES AquaNet 2017 Sampling and laboratory protocols 1. Summary of sampling parameters Table 1 lists all the parameters that will be recorded during the SITES AquaNet pilot experiments These variables will be recorded in each individual mesocosm and in the lake. The variables with an * will be analyzed at Erken Laboratory. Temperature, DO, chlorophyll, phycocyanin and turbidity fluorescence will be recorded with sensors full time deployed in the mesocosms at a 0.6 m depth (Figure 1-2). PAR will also be recorded with sensors at 0.4m depth (except at Svartberget at a 0.2m depth due to the high humic content; Figure 1-2). ph and conductivity, as well as additional DO, temperature and chlorophyll measurements, will be recorded during sampling days (every 2-4 days) with a multiprobe at a 0.6 m depth. PAR will also be recorded during sampling days at three depths (0.05, 0.4 and 0.8 m; except at 0.05, 0.1 and 0.2m at Svartberget) with a sensor with handheld display to determine the light coefficient of extinction. Figure 1. Sensor installations within the mesocosms, from above (left scheme) and from the west (right scheme). Figure source: Ola Langvall (Asa Research Station) Figure 2. Principle construction of the experimental, as seen from above, with 20 mesocosms, four were located in each pool. Only mesocosms 1-16 were used in the experiments Four different treatments with four replicates each were applied in a Latin square design; control (C), fish predation (F), light reduction (L) and combined fish predation + light reduction (FL). A surveillance camera was located in the upper right corner of the, on some of the sites. Figure source: Ola Langvall (Asa Research Station)

2 Total nutrients, dissolved nutrients, particulate nutrients, dissolved organic carbon, DNA, chlorophyll-a, bacteria, phytoplankton and zooplankton sub-s will be obtained from an integrated water column taken with a Ruttner r. Note that collection for particulate nutrient analyses is optional (see below). Periphyton growth in the mesocosms will be monitored through the collection of biomass in PE strips at the end of the experiments. Similar sensor measurements and collection will be done for the lakes, except for the periphyton. Furthermore, one extra PAR sensor (in addition to the one located at 0.4 m; 0.2m for Svarterbeget) will be deployed in the each local lake at a 0.05m depth to continuously obtain the light coefficient of extinction. All stations should also record incoming light in the lakes with a PAR sensor placed above water. Table 1: Sampling parameters recorded in the SITES AquaNet 2017 experiments Parameters Sampling type Location in mesocosms Lake Frequency Quantity Temperature DO PAR Chla (fluor.) Phycocyanin (fluor.) Turbidity (fluor.) ph EC Total nutrients* Dissolved nutrients* Particulate phosphorous (optional)* Particulate nitrogen and carbon (optional)* DOC* DNA* Chla/ spectrophotometry* Aanderaa Oxygen optode 4531 sensor Aanderaa Oxygen optode 4531 sensor Apogee PAR SQ-500 sensor CTG Trilux sensor CTG Trilux sensor CTG Trilux sensor AP-2000 Aquaread multiprobe (handheld) AP-2000 Aquaread multiprobe (handheld) 0.4 m depth, northern Western Western Western Western Western Western Western southern of southern of 0.4 m depth (0.2m at Svartberget), southern of southern of southern of southern of southern of southern of Southern of Southern of Southern of Southern of Southern of Southern of Southern of Continuously & additionally on every sampling day (handheld AP-2000 multiprobe) Continuously & additionally on every sampling day (handheld AP-2000 multiprobe) Continuously & additionally on every sampling day (Apogee MQ-500) Continuously & additionally on every sampling day (handheld AP-2000 multiprobe) Continuously Continuously ml ml From 250 to 1000 ml depending on trophic state From 250 to 1000 ml depending on trophic state 25 ml From 50 to 1000 ml depending on lake trophic state From 100 to 1000 ml depending on lake trophic state

3 Bacteria / flow cytometer* Phytoplankton / microscope* Zooplankton / microscope* Additional water * From a 10L integrated From a 10L integrated Western Western Western Western Periphyton* PE strips Northern 2. Sample collection during experiments Southern of Southern of Southern of Southern of Sample collected on last sampling day 10 ml 100 ml 5 L concentrated in 100 ml ml 2.1. Integrated water collection in mesocosms and lake - Tablet with tagtags application. - Notebook and pencil L-Ruttner r - 15L-buckets (as many as number of people sampling x 2) - Plastic stirrer (as many as number of people sampling) L PE-bottles for sampling (#17) - 100mL-flasks for phytoplankton (#17) - 100mL-flasks for zooplankton (#17) - 1L-plastic jags (as many as number of people sampling) - 65µm-plankton-net (as many as number of people sampling) - 100mm-Ø-funnels (as many as number of people sampling) - 500mL-wash-bottles filled with tap water (as many as number of people sampling) - 2L-PE-bottle filled with tape water - 100mL-glass-bottle with plastic pipette filled with lugols solution - Boxes/bags for transporting bottles/flasks - Large plastic box for transporting material - Cooling boxes - Make sure the tablet (or phone) with the tagtags application is fully charged before leaving to the lake. Bring also a notebook and pencil - this will always be our back up for taking notes - Make sure you cleaned the 2L-PE bottles in the laboratory prior to sampling. Rinse all the bottles several times with demineralized water and let them dry. Avoid having the in of the bottle in touch with anything to prevent contamination or using soapy products to clean them. Use the same bottle (labelled) for each individual mesocosm and lake for future samplings. - Make sure the bucket and r are well cleaned with tap water before bringing them to the lake. - Label the 100mL flasks prior to sampling accordingly (see section below Labeling Samples ). - Add drops of lugols solution to the phytoplankton and zooplankton flasks in the laboratory prior to sampling. Use gloves and work in a well-ventilated area. - Do not handle any food, drink, sunscreen or insect repellant until after the samplings. - Sampling should always start at around a.m.

4 1. When you arrive at the jetfloat fill in the check list in the tagtags application. 2. Take four s with a 2.5L Ruttner r at 0-4m (two s) and m (two s) on the west of the mesocosms. Avoid sampling very close to the edges to reduce wall effects to minimal, as well as close to the sensors. 3. Collect all the water in the same bucket. - Always mix the total water volume in the bucket using a plastic stirrer before start taking the subs below. - Rinse the r, buckets and plastic stirrer with lake water in between mesocosms samplings. 4. Water for total nutrients, dissolved nutrients, particulate nutrients, DOC, DNA, chlorophyll and additional analyses (this should be taken the first, as nutrient s are very easily contaminated): i. Mix the water in the bucket with the plastic stirrer and fill the 0.5-2L PE-bottle from the collection bucket. ii. Place the bottle in a cooling box for transportation if your lake is too far from the field station 5. Phytoplankton subs: i. Mix the water in the bucket with the plastic stirrer, collect water with a 1L jag and fill a 100mL flask for phytoplankton analyses. ii. The flasks should already contain drops of lugols solution. iii. Close the bottle, mix and place the flask back in a box/bag for transportation to the lab. 6. Zooplankton subs: i. Mix the water in the bucket with the plastic stirrer and filter 5L using a 1L-plasticjag through a 65µm plankton net. Do this on top of another to collect the filtered water. ii. Flush the animals stuck in the net by using a wash bottle filled with tap water (no distilled water!). Make sure you collect all the animals at the bottom of the net by flushing three times the entire net. You can refill the wash bottle from the 2L-PEbottle filled with tap water when it is empty. iii. Place a funnel on a 100mL flask and collect the water from the plankton mesh by opening the bottom clamp. Flush once more with tape water the mesh to make sure all animals are collect in the bottle. iv. The flasks should already contain drops of lugols solution v. Close the bottle, mix and place the flask back in a box/bag for transportation to the lab. vi. The filtered water collected in a bucket and the remaining one from the collection bucket should be returned to the mesocosms. 7. Repeat 1-6 for the next mesocosms and lake collection Multiprobe and PAR measurements with handheld display - Tablet with tagtags application - Aquaread AP-2000 multiprobe with handheld display - Apogee PAR SQ-500 with handheld display - Notebook and pencil - Electrical tape - Meter

5 - Mark the sensors cables of multiprobe and PAR sensors for recording measurements at 0.6m and at 0.05, 0.4m and 0.8m depth (0.05, 0.1 and 0.2m for Svartberget), respectively. This can be done using electrical tape and a meter prior to the start of the experiment. - Test and calibrate sensors that will be used following the manufacturer's calibration and maintenance procedures. 1. Record the ph, EC, temperature, DO and chlorophyll fluorescence with the AP-2000 multiprobe at a 0.6m depth in each mesocosm (northern ) and lake. This should be done after the integrated water sampling. 2. Record the PAR at 0.05, 0.4 and 0.8m depths (southern ; 0.05, 0.1 and 0.2m for Svartberget) to later estimate the coefficient of extinction. 3. Record the data in the tablet with the tagtags application. A notebook will be used as a backup in case something happens to the tablet Sample handling at the lab Storage of phytoplankton and zooplankton s collected from the field - Phytoplankton and zooplankton s (#34) - Cold and dark room (4-10 C) - Lugols solution 1. Store the phytoplankton and zooplankton s in a cold and dark room/place Total nutrients and additional water s L water s (#17) - Freezer - 50mL sterile falcon tubes (#34) ATTENTION - Label falcon tubes prior to sampling accordingly (see section below Labeling Samples ). - This should be the first one collected from the bottles brought to the lab, as nutrient s are very easily contaminated. 1. Put on the surgical gloves 2. Mix the 0.5-2L PE-bottles carefully before taking a by rotating the bottle 10 times. 3. Pour 40-45mL into a 50mL sterile falcon tube for total nutrient analyses 4. Freeze s at -20 C 5. Repeat 1-4 steps for additional water s.

6 Bacteria s L water s (#17) - Cold and dark room (4-10 C) - 15mL sterile falcon tubes (#17) µl pipette and tips µl pipette and tips - Formaldehyde 37% (filtered) ATTENTION - Formaldehyde is toxic and cancerogenic. You should work on a fume hood and handle the tubes with care. - Label falcon tubes prior to sampling accordingly (see section below Labeling Samples ). 1. Put on the surgical gloves 2. Mix the 0.5-2L PE-bottles carefully before taking a by rotating the bottle 10 times. 3. Pipette 10mL of water in a 15mL-falcon tube. 4. Fix the with 0.5mL of formaldehyde (final formaldehyde conc. 2 %) 5. Close the tubes and shake the s well. 6. Store s in cold (4-10 C) and dark DNA s L PE-bottles with water s (#17) - Filtration units (#2-6) - 1L-glass-bottles (#2-6) - Hoses - Membrane filters (0.2µm pore size, 47mm Ø) (#17) - 2mL-sterile-tubes (#17) - Measuring-cylinders (#2-6) - Vacuum pump - Surgical gloves (non-powered) - Cryobox - Tweezers - 70% ethanol - Demineralized water - Bench film - Notebook and pencil. ATTENTION - Make sure to keep clean the tweezers and working space (bench and protective film) with 70% ethanol. - Label the 2mL-sterile eppendorf tubes prior to sampling accordingly (see section below Labeling Samples ). 1. Put on the surgical gloves. 2. Mount each filtration unit with one 1L-glass-bottle.

7 3. Take the filters with tweezers and place them onto the filter holders. 4. Connect the pump to the filtration units with the hoses. 5. Turn on the pump (no more than -300 mbar). 6. Mix the 0.5-2L PE-bottles carefully before taking a by rotating the bottle 10 times. 7. Pour water from the 0.5-2L PE-bottles onto the measuring cylinders. 8. Note the volume on the label and a notebook. The volume to be filtered will depend on the trophic status of the lake ml for eutrophic or humic waters and up to 1000 ml for oligotrophic water. You should avoid clogging the filters in order to speed up the filtration. 9. Pour the water from the measuring cylinders onto the filtration units. 10. Let the water flow through until the filter is completely dry. 11. Remove the filters with the sterile tweezers while the vacuum pressure is still on. Fold them inwards and put them into the 2mL-sterile eppendorf tubes. 12. Place the tubes with the filters in a cryobox and store at -20 C. These will be our s for DNA analyses. 13. Wash the filter holders, bottles, columns and measuring cylinders with demineralized water in between filtrations. 14. Repeat steps 1-15 for as many s collected Chlorophyll s L PE-bottles with the water s (#17) - Filtration units (#2-6) - 1L-glass-bottles (#2-6) - Hoses - GF/C filers 47 mm (#17) - 2mL-sterile-eppendorf tubes (#17) - Measuring-cylinder (#2-6) - Vacuum pump - Surgical gloves (non-powered) - Cryobox - Tweezers - 70% ethanol - Demineralized water - Bench film - Notebook and pencil. 1. Repeat the same procedure as for the DNA s, except that you use GF/C filters. Here you may filter more volume, given the larger pore size of GF/C filters Particulate nutrient s L PE-bottles with the water s (#17) - 25mm pre-combusted GF/C filters (#34) - 2mL-sterile-eppendorf tubes (#34) - Multi-filtration unit - Tweezers - 70% ethanol

8 - Beaker - Demineralized water - Cryobox - Freezer - Bench film - Notebook and pencil - Make sure to keep clean the tweezers and working space (bench and protective film) with 70% ethanol. - Pre-combust the GF/C filters at 520 C during 4h prior to the filtration. Keep them in a sterile 50mL falcon tube. - Label the 2mL-sterile eppendorf tubes prior to sampling accordingly (see section below Labeling Samples ). 1. Put on the surgical gloves. 2. Place as many pre-combusted 25mm GF/C filters as you can on the multi-filtration unit. You should do it using clean tweezers on a cleaned bench. Avoid having the filters in touch with any surface to avoid any potential nutrient contamination. Make sure the multi-filtration unit is clean by flushing demineralized water through its columns. 3. Mix the 0.5-2L PE-bottles carefully before taking a by rotating the bottle 10 times. 4. Filter your water s for as many s as your filtration unit allows. Note the volume on the label and notebook. The volume will depend on the trophic status of the lake. You should optimize the filtration by testing some s from your lake first you should then be able to identify approximately how much volume you can filter prior to clogging. Volumes filtered usually range between ml for eutrophic waters and up to 1000 ml for oligotrophic waters. 5. Collect the filter with the tweezers, fold it inwards and place in a 2mL-sterile-eppendorf tube. Place the tube with the filter in a cryobox and store at -20 C. This will be our for particulate N and C analyses. You can discard the filtered water. 6. Repeat steps 2-5 for as many water s collected. 7. Repeat steps 2-5 to collect another filter per mesocosm and lake for particulate P analyses. 8. Store a few pre-combusted 25mm GF/C filters in 2mL-sterile-eppendorf tubes. Place them in the cryobox and store at -20 C. These filters should not have been used for any filtration and will be our controls for estimating the amount of nutrients that the filters contain Dissolved organic carbon and dissolved nutrient s L-PE-bottles with the water s (#17) - 50mL-syringe µm-PES-syringes-filters (#18) - 50-mL-falcon tubes (#34) - Tweezers - 70% ethanol - Freezer - Bench film - Beaker - Demineralized water

9 - Make sure to keep clean the tweezers and working space (bench and film) with 75% ethanol. - Label the 50-mL-falcon tubes prior to sampling accordingly (see section below Labeling Samples ). 1. Put on the surgical gloves. 2. Pour demineralized water in a cleaned beaker and use the water to wash the syringe filter three times with the syringe 3. Mix a 0.5-2L PE-bottle carefully before taking a by rotating the bottle 10 times. 4. Take a few milliliters of water from the 0.5-2L-PE-bottles and wash the syringe filter once more. This is done to avoid the dilution effect created by the previous cleaning step with demineralized water. 5. Filter 40mL of your and collect the filtered water in a 50mL-falcon tube. This will be our for dissolved nutrients. Freeze at -20 C. 6. Repeat steps 2-5 to collect another a 12mL on a 15mL falcon tube for dissolved organic carbon analyses. 7. Repeat steps 2-6 for as many water s collected Periphyton s - 50mL falcon tubes (#16) - Algae scrapper or similar - Label the 50-mL-falcon tubes prior to sampling accordingly (see section below Labeling Samples ). - A wash bottle with tape water (no distilled water!) may help in collecting the biomass from the strip, but you should try not to exceed the 50mL volume in the tube. 1. Locate in northern of each mesocosms a PE strip (length: 1475mm, width: 50 mm, thickness: 3mm). 2. At the end of the experiment retrieve the strip from the mesocosms gently. 3. Collect the biomass attached to the strip on a 50mL sterile falcon tube by using an algae scrapper or similar tool. 4. Freeze the s at -20 C. 3. Labels, storage of s and shipping of s Labels were created for total nutrient s, dissolved nutrients, particulate phosphorous, particulate carbon and nitrogen, dissolved organic carbon, DNA, chlorophyll-a, bacteria, phytoplankton, zooplankton, additional water and periphyton s (see excel files at dropbox folder SITES AquaNet, Labels_Experiments_2017 ). Each label contains critical information for identification during analyses (Fig. 3). All labels are to be printed and attached to collection flasks/tubes well ahead before samplings. To facilitate the organization of analyses later on, it is highly recommended to store and ship the s in the same way as these will be analyzed later in the laboratory. Technicians from each of the stations should then communicate with the technicians in charge of conducting the s analyses

10 before collection and storage (see Table 2). For these experiments, you should separate the 17 s of each sampling point by using a larger recipient labeled with the sampling point, experiment number and lake. Table 2: Environmental parameters analyzed during the experimental tests conducted in summer 2017 Variables Lake(s) # samplings Method Main responsible Laboratory Total phosphorous SS-EN ISO 6878:2005 on a Hitachi U2910 spektrofotometer Helena Enderskog / Margarita judina Erken Laboratory Total nitrogen Shimadzu TOC-L TNM-L Total organic carbon Shimadzu TOC-L TNM-L Dissolved organic carbon Initial and final sampling experiment I and II Shimadzu TOC-L TNM-L Dissolved anions (F, Cl, NO 3, NO 2, PO 4 and SO 4) and cations (Na, NH 4, K, Ca, Mg) Metrohm Ion Chromatography system (883 Basic IC Plus) Particulate nitrogen Erken Costech Elemental Combustion System ECS 4010 Particulate carbon Erken Costech Elemental Combustion System ECS 4010 Particulate phosphorous Erken SS-EN ISO 6878:2005 on a Hitachi U2910 spektrofotometer Helena Enderskog / Margarita judina Erken Laboratory Chlorophyll-a SS , (1) mod on a Hitachi U2910 spektrofotometer Helena Enderskog / Margarita judina Erken Laboratory Bacteria abundance Flow cytometry Bacteria composition Illumina sequencing 16S-based amplification Yinghua Zha Phytoplankton composition Phytoplankton composition and biomasses Zooplankton composition and biomasses PAR, chl-a fluorescence, phycocyanin, turbidity, DO and temperature ph, conductivity and PAR profiling Final sampling experiment I and I. Ccmplete for Erken experiment II Uncomplete datasets experiment I, continuous datasets experiment II Illumina sequencing 18S-based amplification Inverted microscope counts Inverted microscope counts Autonomous sensor system Yinghua Zha Sophie Wertek / Helmut Hillebrand Eglė Petkevičiūtė / Pablo Urrutia Cordero Peder Blomkvist, William Colom/Don Pierson, Bengt Liljeblad, Juha Rankinen and Ola Langvall Oldenburg University Aquatic Ecology/Lund University and Handheld sensor units Field technicians

11 Figure 3. Example labels (Total nutrient s, experiment 1, Svartberget station, sampling 0 before experimental manipulations were carried out, mesocosms 1-4 encompassing 1 replicate from each of the control, fish, light and fish+light treatments) with explanations of the terminology used during the SITES AquaNet experiments in 2017.