2.1. Lake, H2O2 Treatment and Sampling Information

Lake Oosterduinse Meer is located in a rural area close to the Dutch coast (52°16′55″ N, 4°30′28″ E) (Figure 1A). It covers 30 ha, has an average depth of 7 m (maximum depth of 13 m) and is stratified during summer months. The lake is connected with the surrounding flower fields with a canal system. Consequently, the lake water is hypertrophic and especially rich in phosphorus, making it an excellent environment for cyanobacterial blooms.

In 2018, lake Oosterduinse Meer was treated twice with H₂O₂; the first treatment took place on 19 June, the second treatment—on 7 August (Figure 1B). During each treatment, H₂O₂ was applied to the top 5 m of the entire lake using a specialized boat (Figure 1C), carefully adding 4.2 mL of a 598 g L⁻¹ H₂O₂ (50% H₂O₂ (w/w)) stock per 1 m³ of the lake water to treat the lake with a target concentration of ~2.5 mg L⁻¹ H₂O₂. During the June treatment, the boat first treated the shallower waters along the shore of the lake, then went to the lake center, treating the sampling location by spiraling three times around it, before covering the rest of the lake from south to north with 75 diagonal lanes (Figure S1A). The average distance between the lanes was 13.3 m and the net treatment time was 7.3 h. During the August treatment, the boat navigated the lake from south to north with 91 diagonal lanes, while the shallower parts along the shore were treated in between (Figure 2). The average distance between the lanes was 10.9 m and the net treatment time was 9.2 h.

H₂O₂ treatment of the lake. The graph shows (A) the boat track during the treatment in August, (B) the sampling locations and the sampling depth where H₂O₂ concentrations were monitored during the treatment. (C–E) H₂O₂ concentrations measured at different time points after the treatment boat had passed the sampling locations, (C) at 0–1-m depth, (D) at 2–3-m depth and (E) at the 4–5-m depth. Colors in panel (A) indicate the time of day during which a certain section of the lake was treated; different symbols in panel (B) indicate the sampling depth; lines in panels (C–E) are moving averages with a window size of 60 min.

During the treatments, continuous measurements of temperature, O₂ saturation, pH and light intensity were collected using a water column (surface to the bottom) from the center of the lake using a Hydrolab Datasonde 5 (OTT Messtechnik GmbH and Co., Kempten, Germany). Weather data from the weather station Schiphol located ~20 km east of the lake were provided by the Royal Netherlands Meteorological Institute (KNMI) (www.knmi.nl; accessed on 10 January 2020).

Samples for DNA extraction, phytoplankton identification and quantification, analyses of nutrient concentrations and bacterial enumeration were taken from the 0-m and 5-m depth at the sampling location in the middle of the lake. The samples from the 5-m depth were pumped up with a 1.4-bar water pump, type 088 (Barwig Wasserversorgung, Bad Karlshafen, Germany), connected to a 5-m-long tube. One day before the treatment (t = −24 h), seven biological replicates were sampled from the 0-m depth, six biological replicates—from the 5-m depth. For all the other time points, at 0 h (just before the treatment, only for phytoplankton and nutrient analysis), 5 h, 24 h, 48 h and 96 h after the treatment, four biological replicates were taken from both depths. All the samples were collected in individual 5-L transparent plastic bags (DaklaPack, Lelystad, The Netherlands) and immediately processed on the shore.

For DNA extraction, a 1-L subsample was filtered instantly through a 5-µm polycarbonate Cyclopore filter (Whatman GmbH, Dassel, Germany) on a Nalgene™ filtration unit (ThermoScientific, Waltham, MA, USA) to remove most filamentous cyanobacteria, bigger eukaryotic organisms and particles. The filtrate containing planktonic bacteria that were not attached to bigger particles was collected and filtered again over a 0.2-µm polycarbonate Cyclopore filter (Whatman GmbH, Dassel, Germany). This filter was then carefully folded, placed into a 1.5-mL screw cap tube and immediately snap frozen in a CX100 dry shipper (Taylor-Wharton/Worthington Industries, Columbus, OH, USA) at −190 °C and later stored at −80 °C until analysis. Only 0.2-µm polycarbonate Cyclopore filters were used for DNA extraction.

For phytoplankton analysis, 30-mL subsamples were taken from three of the biological replicates, fixed with 600 µL acidic Lugol’s iodine and immediately stored at 4 °C until further analysis.

For bacterial enumeration using flow cytometry, a 1.5-mL subsample was taken from each replicate, fixed with glutaraldehyde (0.5% (v/v) final concentration prepared from a 25% (v/v) EM grade stock solution; VWR, Amsterdam, The Netherlands) at 4 °C for 15 min, after which it was snap frozen and later stored at −80 °C until analysis.

For the quantification of dissolved inorganic nutrients, 55-mL subsamples from each biological replicate were filtered over GF/C filters on a Millipore 1225 Sampling Manifold (Merck KGaA, Darmstadt, Germany). Subsequently, 15 mL of each filtrate were frozen and stored at −20 °C for nutrient analysis.