4.3. Sample Analysis

All Environment Agency incident response algal samples were analysed at the EA biology laboratories by trained and quality assured analysts using high powered microscopes and Sedgewick Rafter counting cell slides. Samples were mixed before analysis to allow even distribution of cells. If the analysis found any nuisance algae genus present, cell counts were performed and compared with warning thresholds for cyanobacteria. Where a scum was present no count was needed and the sample was considered above threshold. The warning threshold levels used were consistent with guidance levels derived from the WHO guidance [34] and modified by [32]. For cyanobacteria blooms the threshold was 20,000 cells/mL. The routine monitoring and commercial water samples were not subjected to microscopy analysis. Cyanobacterial identification was performed using a variety of identification keys, but predominantly with guidance listed in laboratory protocol documents [33]. Identification was performed against a comprehensive database published by the Natural History Museum, containing 5279 species of freshwater algae of relevance to the British Isles.

Water samples received for testing were shaken to mix thoroughly before measuring into a graduated centrifuge tube. Typically, 45 mL sample was taken, unless lower volumes were supplied. Samples were first centrifuged at 4500 rpm for 10 min. If there was no evidence for floating cells, then the majority of the supernatant was decanted from the algal pellet, leaving approximately 1 mL behind in the centrifuge tube. A minimum of 1.5 mL of decanted supernatant was filtered through a 0.2 μm syringe filter directly into a LC-MS glass autosampler vial for extra-cellular toxin analysis. The pellet and approximately 1 mL remaining supernatant was subsequently mixed, and quantitatively transferred to a 2 mL Eppendorf tube. Samples were centrifuged at 13,000 rpm for 10 min, after which the supernatant was removed and discarded. Eppendorf tubes containing the remaining pellets were placed into a freezer (<−15 °C) for a minimum of 30 min to help lyse the bacterial cells. After this time, tubes were removed from the freezer and 1.0 mL of 80% MeOH added. Tubes were vortex mixed for 30 s, before leaving for 30 min. After a further 30 s vortex mix, tubes were again centrifuged (13,000 rpm, 10 min) after which the supernatant was filtered through a 0.2 μm syringe filter and transferred to a glass autosampler vial for intra-cellular toxin analysis.

For bloom samples containing visibly floating cells, a filtration method was used in preference to the centrifugation method. Recorded volumes of well-mixed water samples were filtered through a 0.2 μm Teflon filter using a vacuum pump. The filter was removed and placed into a 50 mL centrifuge tube, before placing in a freezer (<−15°C) for a minimum of 30 min. After removing from the freezer, 5.0 mL of 80% MeOH was added and the tube shaken gently to dissolve the intracellular toxins. Filters were left in solvent for 60 min, with occasional mixing, before filtering through a 0.2 μm syringe filter into a glass autosampler vial for intra-cellular toxin analysis.

Chemical analysis of cyanotoxins was conducted as detailed in [40]. A Waters (Manchester, UK) Acquity UHPLC system coupled to a Waters Xevo TQ tandem quadrupole mass spectrometer (MS/MS) was used with a 1.7 μm, 2.1 × 50 mm Waters Acquity UPLC BEH C18 column in conjunction with a Waters BEH C18 guard cartridge. The column was held at +60 °C, and a 5 μL injection volume utilized, together with mobile phase flow rate of 0.6 mL/min. Mobile phase A1 consisted of water +0.025% formic acid, mobile phase B1 comprised acetonitrile (MeCN) +0.025% formic acid. The UHPLC gradient started at 98% A1, dropping to 75% A1 at 0.5 min holding until 1.5 min, dropping further to 60% A1 at 3.0 min, decreasing further to 50% A1 at 4 min, before a sharp drop to 5% A1 at 4.1 min, holding until 4.5 min before increasing back to 98% A1 for column equilibration at 5 min for a further 0.5 min. Each instrumental sequence started with a series of instrumental blanks, followed by toxin calibration standards and a microcystin chromatographic retention time marker solution. The MS/MS source parameters and Selected Reaction Monitoring (SRM) transitions were exactly as specified in [40]. Quantitation of microcystins was performed against external calibration standards with results calculated in terms of μg/L of water.