2.2. Microcosm Design and Sampling

Microcosms were created based on ten treatments (Supplementary Materials Table S1) using sterile 40 mL glass vials with PTFE-lined silicon septa. Treatments were destructively sampled in triplicate over a series of time points (1, 3, 7, 14, 21 days), ensuring samples were temporally independent. Each 40 mL microcosm (excluding seawater only) contained 20 mL of seawater which was supplemented with nutrients (final concentrations of 300 µM NH₄Cl and 20 µM K₂HPO₄); this allowed us to control for nutrient limitation and evaluate microbial response to oil/dispersants in the early phase. The sampling site is subjected to large annual inputs of dissolved inorganic nitrogen (DIN) and dissolved inorganic phosphorous (DIP) from the river Thames [44] and the nutrient loadings are representative of background nitrogen and phosphorous concentrations recorded at the sampling site at other times [45]. The oil (0.1% v/v final concentration) was a Norwegian Geochemical Standard, North Sea Oil (NSO-1; Norwegian Petroleum Directorate, Stavanger, Norway), that had previously been weathered (distilled at 69 °C, which is the boiling point of hexane and thus removes the most volatile components of crude oil). Oil was added to microcosms by reverse-pipetting and time-zero samples were analysed to ensure consistent oil loadings, which consistently added 45.50 and 4.38 µg mL⁻¹ ± 7% of resolvable alkanes and PAHs, respectively. The dispersants were Slickgone NS, Superdispersant 25, and Finasol OSR 52 added to an industry standard (0.005% v/v), creating a ratio of 20:1 oil to dispersant [46]. The biosurfactants were rhamnolipid, sophorolipid, and trehalolipid, and were also added at a final concentration of 0.005% v/v to create the same ratio, based on the percentage of active ingredient. Killed controls (seawater and oil microcosms with 1.1049 mM of added HgCl₂) were also sampled at each time point to monitor abiotic hydrocarbon loss. All microcosms were incubated on a rotary shaker at 100 RPM at 16 °C, replicating summer environmental conditions in the Thames Estuary.