Supplement to Stoecker et al. “Underestimation of microzooplankton grazing in dilution
experiments due to inhibition of phytoplankton growth”
Methods for Determination, Analysis and Quantification of particulate and dissolved PUAs
Determination of the production of PUAs by cells was performed according to a slightly
modified protocol based on Vidoudez et al. (2011). Important steps in the Vidoudez et al.
protocol are briefly described, whereas steps which varied from the Vidoudez et al. (2011)
protocol are described in detail.
For particulate PUA, 1 to 5 L of mesocosm water depending on cell densities was
concentrated on a GF/C filter (Whatman, Dassel, Germany) under vacuum (min. 500 mbar). This
protocol was verified not to result in the release of PUAs from cells. The filter was rinsed with
1.5 mL of a 25 mM O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA,
Roth, Karlsruhe, Germany) solution in 100 mM TRIS-HCl, pH 7.2. The filter and the cell
suspension were transferred into a glass vial and were mixed on a vortexer, 5 µL of the internal
standard (benzaldehyde 1 nM in methanol, Sigma Aldrich) was added, the vial was closed and
shaken. The samples were frozen and thawed for mechanical cell disruption, incubated and
subsesquently stored according to the original protocol (Vidoudez et al. 2011). PUA extraction
was performed according to Vidoudez et al. (2011), but, for the first extraction step, glass
spheres were added and the suspension was vortexed for 2 min. after the addition of the 750 µL
methanol and 1.5 mL hexane. Finally, the hexane extracts were vacuum dried and re-dissolved in
80 µL hexane for GC-MS measurements.
For dissolved PUAs, 5 µL of internal standard (benzaldehyde, 1 mM in methanol) was
added to 1 L of the mesocosm water in a PP bottle. The samples were filtrated on a GF/C filter
(Whatman, Dassel, Germany) under vacuum (max. 500 mbar) and the filtrate was transferred
through teflon tubing with a flow rate of circa 1 L h-1 to a 3 mL EASY® solid phase extraction
cartridge (Macherey-Nagel, Düren, Germany) which had been pretreated with 1 mL of a PFBHA
solution in 100 mM TRIS-HCl, pH 7.2. The liquid retained in the cartridge was reduced by
applying overpressure from an empty syringe to the cartridge. EASY® cartridges were then
washed with 2 x 2 mL deionized water, dried in vacuum (circa 600 mbar) and eluted into glass
vials with 2 x 2 mL of a 5 mM solution of PFBHA in methanol. After incubation for 1 h at room
temperature, samples were stored at -20°C. The PUA extraction was accomplished according to
Vidoudez et al. (2011b). Finally, the hexane extracts were vacuum dried and re-dissolved in
80 µL hexane for GC-MS measurements. The identical protocol without the GF/C filtration step
was followed for the determination of the purification success of charcoal cartridges (see below).
The samples were measured with GC/MS (ISQ Trace GC Ultra, Thermo Fisher, Dreieich,
Germany) equipped with a 0.25 mm x 30 m DB-5MS GC column (Agilent,
Böblingen, Germany) in electron impact (EI 70 eV) mode. Data were evaluated with the
Xcalibur Quan Browser (2.1.0 SP1.1160). Identification of PUAs was based on the retention
time compared with standards (except octatrienal that was identified based on its characteristic
mass spectrum) and major fragment ions as described in Vidoudez et al. (2011). The
quantification was based on the ratio between the molecular ions of the derivatized PUAs (m/z
319 for octadienal, 317 for octatrienal, 347 for decadienal) or the major fragment m/z 276
(present in all measured PUAs) for octadienal and the fragment m/z 271 of the derivatized
internal standard. The following quantification standards were produced and derivatized, 0.5, 1,
2, 5, 10, 20, 50, 75 nM from heptadienal (<97%, Sigma-Aldrich), octadienal (96+%, SigmaAldrich) and decadienal (85%, Sigma-Aldrich) according to the original protocol (Vidoudez et
al. 2011). The same calibration as for octadienal was used for octatrienal. The selected
quantification curves for each standard were dependent on the detection limit of each PUA and
the maximal amount found in the sample with the highest concentration. The GC temperature
program for the separation was 60°C (held for 2 min) then ramped with a rate of 8°C min-1 to
240°C and then with a rate of 15°C min-1 to 300°C (held for 3 min).
Reference
Vidoudez C., R. Cascotti, M. Bastianini, and G. Pohnert. 2011. Quantification of dissolved and
particulate polyunsaturated aldehydes in the Adriatic Sea. Marine Drugs 9: 500-513
doi:10.3390/md9040500.