Outline of the experiment:
Filtering varying sampling volumes of seawater through 0.4 µm filters, with each sample volume processed in triplicates.
Experimental volumes: 1000 mL x 3, 500 mL x 3, 200 mL x 3, 20 mL x 3 + negative control: 500 mL x 3 of distilled water (dH2O).
Additionally, 100 mL of the same seawater is required for phytoplankton morphology-based analysis.
In total, about 5,500 mL of seawater and about 1,700 mL of dH2O is needed.
Items provided:
0.4 µm filters (Whatman, product nr. 10417712): packaged separately for each experimental volume and negative control, four filters per pack (one filter added as a backup per volume);
1.5 mL tubes: three tubes per sample volume + negative control. Colors: white: 500 mL of dH2O, green: 20 mL, blue: 200 mL, yellow 500 mL, red: 1000 mL;
50 mL tube: used for measuring 20 mL volume;
Marker pen to label tubes.
Items needed for sampling and filtration from each participant (not provided):
70% ethanol for cleaning surfaces and for the alcohol burner (if used);
Paper towels to clean surfaces;
Lab gloves;
10% HCl or bleach solution for sterilizing the sampling flasks/jugs and carboy;
Sampling flasks/jugs to collect the water;
Carboy for pooling all the water needed and for mixing;
Filtration system(s) and pump(s). The filtration system(s) should be autoclaved or acid-washed prior to use;
Tweezers to handle filters. The tweezers should be sterilized before usage and between using another sample volume;
Alcohol burner or Bunsen burner and matches to sterilize the tweezers between filtering samples;
Graduated cylinder for measuring sample volume;
Lugol’s solution to fix phytoplankton samples;
100 mL glass bottle for phytoplankton sample storage;
dH20 for negative controls.
Sampling protocol
Collect the required volume of seawater needed for the experiment (filtration + phytoplankton) from the same depth used for routine phytoplankton sampling (surface layer/ integrated sample of 0–10 m);
Use clean sampling flasks/jugs to collect the seawater (acid-washed: 10% HCl or store-bought bleach solution) and well-rinsed: 10 x with tap water, 3 x with dH20).
Pool all collected water in a clean carboy (cleaned as above) and mix well;
Take 100 mL of the water and fix it with Lugol’s solution;
The sample water can be stored for a few hours at the ambient temperature if it is similar to the water temperature or in the fridge/climate room at 4 °C. Please take note of the time that the sample was stored before filtration.
Filtration protocol:
Clean the surface used for filtration with ethanol;
Thoroughly mix the seawater in the carboy before taking each sample volume and wear gloves when handling samples, filters, tweezers, and tubes;
Begin with filtering the negative control: filter 500 mL of dH2O through a 0.4 µm filter;
The filtration time per filter should be within 60 min and the pressure should not exceed 200 mm Hg/ 267 mbar / 27 kPa. Note any deviations in time/pressure;
Once the filtration is finished, fold the filter with sterile tweezers several times to fit into a 1.5 mL tube;
Label tubes using the provided format (first two letters of the institute/university+A/B/C for each replicate and 1–4 for each volume starting from dH2O, e.g. for TalTech dH2O: TAA1, TAB1, TAC1 (please see the “Sample information sheet.xlsx” for example);
We will know the sample volume based on the coloured sticker on the tube (please see under the items provided). If you wish you may write down the sample volume on the tube;
Please store the filter immediately after inserting it into a 1.5 mL tube at -20 °C;
Continue with replicates of the same sample by filtering each of those on a separate 0.4 µm filter as mentioned above;
After the filtration of negative control in triplicates is finished, continue with the smallest sample volume (20 mL) and proceed to the next largest volume until all the volumes have been filtered in triplicates.
Phytoplankton analysis:
Please analyse the phytoplankton sample using the same approach as for routine monitoring in your country.