Gf f membrane

Sampling was carried out by performing three surveys of the SYS (33–36 °N, 120–124 °E) in 2018 onboard R/V KEXUESANHAO for 11, 16, and 16 sampling sites on April 18 to 22 (spring), August 29 to September 6 (summer), and November 19 to 27 (autumn), respectively (Fig. 1).
Zooplankton samples were collected by vertical tows using a WP2 plankton net (mouth area: 0.25 m², mesh size: 200 μm) from about 3 m above the bottom to the surface. The collected samples were stored in formalin–seawater solution with a final concentration of 5%. Preserved samples were examined by stereoscopic microscope (SZM-LED2, OPTIKA) to identify the zooplankton community morphologically. In situ temperature and salinity were obtained using a shipboard rosette-mounted Conductivity-Temperature-Depth casts (CTD, Sea Bird 911) with the probe of temperature and conductivity, respectively. About 500 ml seawater was collected from surface and bottom layers, respectively, for chlorophyll a (Chl a) measurement. Seawater was filtered through GF/F membrane (Whatman) and stored in liquid nitrogen until analysis. Chl a concentration was determined in the laboratory using a UV fluorescence spectrophotometer (F-4500, Hitachi, Japan) after extraction with 90% acetone for 24 h under 4 °C (Shi et al. 2018 (link)).

Withdrawn cell suspensions (5 mL) were filtered through a Whatman GF/F membrane. Pigments were extracted in a 10 mL tube with 80% chilled acetone and kept in darkness for 12 h. Then, the tubes were centrifuged at 4 °C for 15 min at 6000× g. The optical densities of the extracts at 646, 663 and 750 nm were determined using a UV-VIS spectrophotometer (UV-1700, Shimadzu). The concentration of Chl a was calculated according to the method from Lichtenthaler and Wellburn [24 (link)].

Triplicate samples of 200–300 mL were filtered through 20 (nylon, Millipore), 2 (polycarbonate, Whatman) and 0.2 (polycarbonate, Whatman) μm aperture sized membranes in time series, and total Chl a concentrations were calculated by combining size-separated Chl a concentrations for the 20 L incubation systems, and 100–150 mL were filtered through a GF/F membrane (Whatman) to measure total Chl a for the 2 L incubation systems. The pigments on the membranes were extracted with 90% acetone at -20 °C for 20 h in the dark. The extract was used for measuring Chl a using a 7200-000 Trilogy fluorometer (Turner Designs)^{47 (link)}.

The concentration of dissolved phenol was determined every two days after filtration (using GF/F membrane, Whatman) based on the standard method [26 (link)]. Phenol concentrations were measured via a spectrophotometer (Shimadzu UV-2450, Japan) after chloroform extraction. The absorbance of the colored complex of phenol with 4- amino antipyrine was detected at 460 nm [26 (link)]. Moreover, to detect the abiotic degradation of phenol during the experiment, a blank control (BG11 medium with phenol and without algal cells) was designed to measure the concentration of phenol every two days.

TOC and DOC were determined using a Shimadzu TOC-L analyzer (Xu et al., 2011b (link)). For POC analysis, water sample was filtered through a pre-combusted GF/F membrane (0.7 μm, Whatman, United States), and then quantified using a Perkin Elmer Series II CHNS 2400 analyzer, after HCl-fuming to remove the carbonates. Acetanilide (71.09%) was used as the analytical standard (Xu et al., 2011a (link)). Samples from the offshore mesocosm were limited, therefore only samples from the coastal mesocosm were analyzed.

The Dunaliella tertiolecta culture was exposed to Control (f/2 medium) and WAF at a cell density of 5 × 10⁴ cells · mL⁻¹ in batch culture for a period of 48 h in triplicates. The role of Kreb's cycle on metabolism during oil exposure was confirmed through determination of respiration rates after 48 h time point. Respiration rates were measured using a Clark‐type oxygen electrode (Hansatech, Norfolk, UK), as per methods described in Kamalanathan et al. (2015 , 2021a (link)). Further confirmation of the upregulation of Kreb's/tricarboxylic acid (TCA) cycle was confirmed by measuring the concentration of cycle metabolites and rates of respiration. Briefly, cells of D. tertiolecta were collected after 48 h incubation in the Control and WAF treatment. For measuring the concentration of metabolites in the cells, 400 mL of cultures were filtered through GF/F membrane (0.7 μm, Whatman, United States) and 0.5 ppm of ¹³C‐citrate was added to the tubes as an internal standard and the metabolites were extracted using chilled 100% methanol. The methanol extract was then analyzed on LC–MS/MS for organic acid analysis.