Whatman gf f filters

After 18 days, periphyton was sampled to analyse chlorophyll a concentration, photosynthetic pigments, photosynthetic activity, community tolerance to Cu and microbial composition of prokaryotes and eukaryotes. For each microcosm, a periphyton slurry was produced by scraping off the periphyton from the slides into 150 mL of sea water, filtered through 0.2 μm and amended with the same amount of nutrients as used in the microcosms. Five milliliters of periphyton slurry were filtered through Whatman GF/F filters and used immediately for chlorophyll a analyses. Ten milliliters of periphyton slurry were filtered through Whatman GF/F filters, frozen at −20°C and stored until pigments extraction. Ten milliliters of periphyton slurry was aliquoted in tubes, pelleted by centrifuged at 6500 g for 10 min at room temperature, the supernatant was removed and the resulting pellets were snap-frozen in liquid nitrogen and stored at −80°C until DNA extraction. The remaining periphyton slurry was used to determine photosynthetic activity (¹⁴C-incorporation) and tolerance measurements following the PICT approach. Analyses of chlorophyll a, pigment profiles and microbial composition were done for all treatments. For logistic reasons, photosynthetic and community tolerance measurements were only done for the control microcosms and the microcosms with a Cu exposure of 0.32 and 1.78 μM Cu.

Salinity, temperature, dissolved oxygen and fluorescence were measured with a Sea-bird electronics (SBE) 911plus CTD and a 10-L SBE 32 rosette water sampler. Seawater for dissolved inorganic nutrients (Nitrate + Nitrite, Ammonia, dissolved reactive phosphorus), particulate organic matter (POC and PON), and chlorophyll a (chl-a) concentration, were sampled from the Niskin using acid-washed silicone tubing. Nutrient samples were filtered through Whatman GF/F filters into clean 250 mL polyethylene bottles and kept at -20°C until analysis using an Astoria Pacific API 300 micro-segmented flow analyser (Astoria-Pacific, Clackamas, OR, United States) according to the colorimetric methods described in (Law et al., 2011 (link)). POC and PON samples were analyzed using an Elementar Vario EL 111 CHN analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) following standard combustion techniques. Samples for chl-a analysis were filtered on-board on Whatman GF/F filters using low vacuum (<200 mm Hg), filters were folded and placed in 1.5 mL cryovials at -80°C until analysis following 90% acetone extraction and spectrofluorometry standard methods.

Physical and biogeochemical parameters were measured at each station. The vertical profiles of temperature and salinity were measured using the CTD (Sea Bird 911plus, Electronics) and density was derived from the same. Nutrient samples (i.e., ammonium, nitrate + nitrite, phosphate (PO₄), and silicate (SiO₂)) were analyzed in the onboard laboratory using a continuous flow auto analyzer (QuAAtro, Seal Analytical, UK). In this study, dissolved inorganic nitrogen (DIN) represents the sum of ammonium, nitrite, and nitrate. Cells for Chl-a analysis were filtered onto 25 mm Whatman GF/F filters, extracted in 90% acetone at 4 °C for 24 hours, and quantified using a Turner Designs fluorometer (Trilogy Fluorometer, Turner Designs, USA).

Aliquots of 10 mL of algal culture were filtered onto Whatman GF/F filters (Whatman, Maidstone, UK) and immediately frozen. Absorption spectrum measurements were performed as previously described, and correction factors (e.g. due to filter absorption enhancement) were applied accordingly [37] . Absorption was measured between 280 and 800 nm with 1-nm increments on a spectrophotometer (Hewlett-Packard HP-8453E) equipped with an integrating sphere RSA-HP-53 (Labsphere Inc., North Sutton, NH, USA). The mean integrated absorption value (a^*) was thus normalized by the chlorophyll (Chl) a concentration to obtain the Chl a-specific absorption coefficient (a^*_ph; m² mg Chl a⁻¹). The number of absorbed photons Chl a⁻¹ integrated over time (expressed in mol photons mg Chl a⁻¹) was calculated as the product of PAR (λ, 400–700 nm) and a^*_ph (λ, 400–700 nm) integrated over the time course of the experiments.

Water samples (1 L) were collected from the six light depths (100%, 50%, 30%, 12%, 5%, and 1% of the PAR) for assessing total and size-fractionated chl-a concentrations. During the three cruises, water samples (0.3 L) were filtered using 25 mm Whatman glass fiber filters (GF/F) for the total chl-a concentration. For the size-fractionated chl-a concentration, water samples (0.7 L) were progressively filtered through Nucleopore filters with pore sizes of 20 µm and 5 µm, and Whatman GF/F filters with pore size of 0.7 µm. The filters were immediately stored in the freezer (−20 °C) until the analysis on board. After a 24 h extraction with 90% acetone, all the chl-a concentrations were quantified using a pre-calibrated Turner Designs model 10-AU fluorometer.

β-Galactosidase and acid phosphatase activities were assayed using chromogenic substrates o-nitrophenyl-β-D-galactopyranoside^{52 (link)} and p-nitrophenylphosphate^{53 (link)}, respectively. Renilla luciferase was assayed using commercial kit (Biotium Inc., cat. #30004-1). The values were normalized to the total cellular protein.
The ³²P_i uptake was assayed according to the previously described protocol^{33 (link)} as follows. Cells cultures grown overnight in YPD were 20-fold diluted with the same medium and grown additionally for 4 h. Cells from 1 ml of obtained cultures were harvested by centrifugation, washed with 3% glucose solution containing 25 mM Tris-succinate buffer pH4.5 or 25 mM Tris-succinate buffer pH8.0 and 10 mM NaCl, and re-suspended in 30 µL of the same buffer. The uptake was initiated by adding 1 µL of [³²P] orthophosphate (2.4 Ci L⁻¹, 50 Ci mmol⁻¹) to the cell suspension. Phosphate uptake was terminated by addition of 1 mL of ice-cold Tris-succinate dilution buffer. The cell suspensions were immediately filtered through the Whatman GF/F filters (Whatman, UK) and washed with the same cold dilution buffers. The radioactivity retained on the filters was determined by liquid scintillation spectrometry.

Primary production (PP) was measured using the ¹⁴C method (Gargas, 1975 ). Five milliliter samples of seawater were added to four 20 mL transparent polycarbonate tubes and mixed with 7.2 μL Na₂¹⁴CO₃ (Centralen Denmark, specific activity = 100 μCi mL^-1). The tubes were incubated in situ for approximately 3 h at 1 m depth, with one of the replicates being incubated in dark. Next, a sub-sample of 5 mL from each tube was mixed with 150 μL 6 M HCl. After 30 min of bubbling, 15 mL scintillation cocktail was added and thoroughly mixed. The samples were analyzed in a scintillation counter (Beckman Coulter LS 6500/Packard Tri-Carb 1600 TR) and net PP was calculated as describe in Andersson et al. (1996) (link).
Samples for chlorophyll-a (Chl-a) were filtered onto Whatman^®GF/F filters, extracted overnight in the dark in 95% ethanol, and then measured on a Perkin Elmer LS 30 spectrofluorometer (Waltham^®, Middlesex, MA, United States) operating at excitation and emission wavelengths of 433 and 673 nm, respectively.