Hpf millex

To investigate the physical-chemical parameters of the collected samples, the samples were processed in the laboratory as early as possible. Prior to the analysis, the samples were kept at 4 °C and were filtered over a 0.45 µm membrane filter (HPF Millex, Merck, the Netherlands). Thiosulfate, sulfate, and sodium were measured by ion chromatography as described by Roman et al. (2015 (link)). Total inorganic carbon was quantified using a TOC-VCPH/CPN analyzer (Shimadzu, The Netherlands). Carbonate and bicarbonate ions concentration were estimated from the total inorganic carbon using the Henderson-Hasselbalch equation (Po and Senozan 2001 (link)) and further used to express the alkalinity of the system as concentration NaHCO₃ (de Rink et al. 2019 (link)). The pH and conductivity of the well-mixed unfiltered samples were measured using the kit from Mettler Toledo, USA.

Plant extraction was performed by accelerated solvent extraction Dionex ASE 350 (Thermo Scientific, Waltham, MA, USA). For all extractions, 100 mL containers were charged with 10–12.5 g plant material mixed with diatomaceous earth (60-033854, Thermo Scientific, Waltham, MA, USA) as a neutral matrix to assure a 1:20 plant/solvent ratio. The ASE extraction was performed with 4 cycles in total, each cycle having a 6 min static time and a 160 s purge time at 1460 psi static pressure. The extraction temperature was 90 °C for all solvents and additionally 30, 70 and 150 °C for ethanol extraction. The obtained extracts were filtered through a 0.22 μm membrane (HPF Millex^®, Merck Millipore, Burlington, MA, USA) and evaporated under vacuum at 60 °C. The extract yield (mg/g dry plant) was calculated using Equation (1) and the fatty acid yield extracted from 1 g plant material (mg/g dry plant) was calculated using Equation (2).