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A total of 369 lakes were sampled using standardized sampling, sampling processing and preserving protocols. Sampling took place during the two warmest weeks for each region: data collectors identified the right sampling period using long term air temperature data spanning at least 10 years. The sampling location was either the historically sampled point, or the centre of the lake, if the lake was not routinely sampled. A temperature profile defined the sampling depth, taken as the bottom of the thermocline. An integrated water column sample was acquired from 0.5 m below the surface till the bottom of the thermocline. This was defined as the point where there was a ≥ 1 degC change of temperature per meter lake depth (-1 degC m-1). If the lake was shallow, then the entire water column was sampled till 0.5 m above the bottom.

A do-it-yourself device, was proposed to data collectors in order to acquire the integrated epilimnetic sample. In brief, a long, transparent and flexible hosepipe of a length dependent on the depth of the sampled lakes was used as the main body of the integrated sampler. The diameter of the hosepipe was appropriate to sample the required volume for all the parameters (ca. 5-10L for hypertrophic and eutrophic, ca. 15 - 30L for mesotrophic and oligotrophic), aiming to obtain the required volume in an ‘acceptable’ number of runs (number of integrated samples). For the construction of the sampler, a weight, anything that was adequate to keep the hosepipe vertical in the water, along with a cord to pull the hosepipe from depth, were connected to the lower end of the hosepipe. Additionally, a rubber stopper was used to firmly close the upper end of the hosepipe and create the required vacuum pressure to hold the integrated water sample in the hosepipe. To obtain the integrated water sample, the hosepipe was lowered with the bottom end open in the water, at a depth of just under the end of the thermocline. When the hosepipe was vertical and the water level was visible at the surface layer of the hosepipe then the stopper was inserted to create vacuum pressure. The bottom end of the hosepipe was pulled to the surface to collect the integrated water sample in a bucket.

All samples were shipped to and stored at the University of Wageningen (The Netherlands) till further analysis. Analyses for pigments, cyanotoxins and nutrients were done centrally in dedicated laboratories in the Netherlands, Germany and Switzerland to minimize analytical errors and maximize integration of the datasets.

Pigment analysis

Samples for pigment analysis were collected on filters, stored in dark and in -20 degC till shipping. The analysis of pigments was modified from the method described by Van der Staay et al. (1992). All filters were freeze dried for 6 hours. Filters (45 mm GF/C and GF/F) were cut in half, placed in separate Eppendorf tubes, and kept on ice until the end of the procedure. 600 µl of 90% acetone were added to each tube along with a small amount of 0.5 mm beads. To release the pigments from the phytoplankton cells, filters were placed on a bead-beater for one minute. Next, they were placed on an ultrasonic bath for ten minutes to increase the extraction yields. This procedure was repeated s twice to ensure a complete extraction of the total pigment content of the filters. To achieve binding of the pigments during the High-Performance Liquid Chromatography (HPLC) analysis, 300 µl of a Tributyl Ammonium Acetate (1.5%) and Ammonium Acetate (7.7%) mix were added to each tube. Lastly, samples were centrifuged at 15,000 rpm and 4degC for ten minutes. 35 µl of the supernatant from both Eppendorf tubes of a filter, were transferred into a HPLC glass vials. Pigments were separated on a Thermo Scientific ODS Hypersil column (250 mm × 3 mm, particle size 5 μm) in a Shimadzu HPLC and using a KONTRON SPD-M2OA diode array detector. The different pigments were identified based on their retention time and absorption spectrum and quantified by means of pigment standards.

Cyanotoxin analysis - Microcystin (MC) analysis

Samples for cyanotoxin analysis were collected on filters and stored in -20 degC till shipping. In the laboratory, frozen filters were transferred to 8 mL glass tubes and freeze-dried (Alpha 1-2 LD, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Hereafter, filters were extracted using hot methanol (75 % MeOH) by adding 2.5 mL 75 % methanol – 25 % ultrapure water (v/v) to each filter, closing the tubes with a screw cap and placing them for half an hour at 60 degC. Subsequently, the extract was transferred to a clean 8 mL glass tube. The procedure was performed three times for each filter; the extracts were joined making a final volume of 7.5 mL. These were dried in a Speedvac (Thermo Scientific Savant SPD121P, Asheville, NC, USA), where after the MCs were reconstituted in 900 μL 100 % MeOH. The reconstituted samples were transferred into 2 ml Eppendorf vials with a 0.22 μm cellulose-acetate filter and centrifuged for 5 min at 16,000×g (VWR Galaxy 16DH, Boxmeer, The Netherlands). Filtrates were transferred to amber glass vials and analyzed for eight MC variants (dm-7-MC-RR, MC-RR, MC-YR, dm-7-MC-LR, MC-LR, MC-LY, MC-LW and MC-LF) and nodularin (NOD). The analysis was done by Liquid Chromatography with tandem Mass Spectrometry detection (LC-MS/MS) as described in [29]. This LC-MS/MS analysis was performed on an Agilent 1200 LC and an Agilent 6410A QQQ (Waldbronn, Germany). The elution program, MS/MS settings of the eight different MC variants and NOD, recoveries, repeatability, limit of detection, and limit of q uantification are given [29]. Each MC variant was quantified against a calibration curve and corrected for recovery. The calibration curves were made using certified calibration standards obtained from DHI LAB Products (Hørsholm, Denmark).

Cyanotoxin analysis - Cylindrospermopsin (CYN) and Anatoxin (ATX) analysis

For the extraction of CYN and ATX 1.5 ml of 0.1% formic acid (FA) was added to the freeze-dried filters, sonicated for 10 min, shaken for 1 hour and then centrifuged. This extraction procedure was repeated two more times and the combined supernatants were dried in a Speedvac (Eppendorf, Germany). Prior to analysis the dried extracts were re-dissolved in 1 ml 0.1% FA and filtered (0.2 µm, PVDF, Whatman, Maidstone, UK).

LC-MS analysis were carried out on an Agilent 2900 series HPLC system (Agilent Technologies, Waldbronn, Germany) coupled to a API 5500 QTrap mass spectrometer (AB Sciex, Framingham, MA, USA) equipped with a turbo-ionspray interface. The extracts were separated using a 5 mm Atlantis C18 (2.1 mm, 150 mm column, Waters, Eschborn, Germany) at 30degC. The mobile phase consisted of water (A) and methanol (B) both containing 0.1% FA, and was delivered as a linear gradient from 1% to 25% B within 5 min at a flow rate of 0.25 mL min-1. The injection volume was 10 µL. Identification of CYN and ATX was performed in the positive MRM mode with the following transitions: CYN m/z 416.1 [M + H]+ to 194 (quantifier) and 416.1/176, and ATX m/z 166.1 [M + H]+ to 149, 166.1/131, 166.1/91, 166.1/43 (quantifier). Mass spectrometric parameters are given in (Fastner et al. 2018) Certified reference standards were purchased from National Research Council (Ottawa, ON, Canada). The detection limit was 0.003 ng on column for CYN and ATX.

Nutrient analysis

Total phosphorus and nitrogen were measured in unfiltered subsamples. Sample bottles were acid washed overnight in 1M HCl and rinsed with demineralized water before usage. Nutrient analyses were performed at the University of Wageningen. Nutrients were measured using a Skalar SAN+ segmented flow analyser (Skalar Analytical BV, Breda, NL) with UV/persulfate digestion integrated in the system. The total phosphate and nitrogen were analysed following Dutch standards protocols (NNI 1986, NNI 1990).

References

van der Staay, G.W.M.; Brouwer, A.; Baard, R.L.; van Mourik F. Separation of photosystems I and II from the oxychlorobacterium (prochlorophyte) Prochlorothrix hollandica and association of Chlb binding antennae with PS II. Biochim Biophys Acta, 1992. 1102, 220-228.

Fastner, J. et al. Fatal Neurotoxicosis in Dogs Associated with Tychoplanktic, Anatoxin-a Producing Tychonema sp. in Mesotrophic Lake Tegel, Berlin. Toxins, 2018. 10.Fastner, J. et al. Fatal Neurotoxicosis in Dogs Associated with Tychoplanktic, Anatoxin-a Producing Tychonema sp. in Mesotrophic Lake Tegel, Berlin. Toxins, 2018. 10.

NNI, Water: photometric determination of the content of dissolved orthophosphate and the total content of phosphorous compounds by continuous flow analysis, in NEN 6663, N.N. Institute, Editor. 1986: Delft The Netherlands (In Dutch).

NNI, Water: photometric determination of the content of ammonium nitrogen and the sum of the contents of ammoniacal and organically bound nitrogen according to Kjeldahl by continuous flow analysis in NEN 6646, N.N. Institute, Editor. 1990: Delft, The Netherlands.