Study area
Legedadi Reservoir, one of the major drinking water supply sources for the capital city of Ethiopia, Addis Ababa, is located approximately 25 km east of Addis Ababa. It is found at an altitude of 2450 m, and geographical position of 9° 01′ N–9° 13′ N latitude and l38° 60′ E–39° 07′ E longitude (Fig. 1).
The reservoir was constructed in 1967 and is administered by Addis Ababa Water and Sewerage Authority (AAWSA). It had an initial storage capacity of 45.9 million cubic meters and surface area of 5.33 km2 [30]. The maximum and mean depths of the reservoir at 2466 meter above sea level are 30 and 4 m, respectively.
The reservoir has an outflow rate (to water treatment plant) of 126,666 (m3/day) and water retention time of 325 days [30]. The catchment of the reservoir has a total area of 205.7 km2. It is a sub-catchment of the Akaki River basin, which flows in a northeast–southwest direction and forms the northwest corner of the Awash River basin. The reservoir's catchment has an annual sediment yield of 845 t/km2/year. Expectedly, the huge sediment transport is accompanied by associated high nutrient load on the reservoir.
Sampling protocol
Two sampling sites were carefully selected considering the areas of frequent surface scum appearance and application of CuSO4 by Addis Ababa water and sanitation authority (AAWSA).
The first site (S1) was located near the dam (deepest part of the reservoir), while the second one (S2) was located at the center of the reservoir. Due to budget limitations, sample collection was conducted only once during the peak bloom month (January 2018) using a Van Dorn bottle sampler Horizontal Model (Eijkelkamp Soil & Water supplier) to document baseline information on the contamination of the reservoir water with cyanotoxins. Samples were taken from the surface and 0.25 m depth and mixed in equal proportions to produce a composite sample. For the identification of major cyanobacterial genera, the composite samples were properly mixed, and 1000 ml aliquots were dispensed into bottles and fixed with Lugol’s iodine (0.01% v/v).
In situ measurements
The transparency of the water (Secchi depth, ZSD) was estimated using a 20-cm-diameter black and white Secchi disk (United Scientific Supplies SCDSK1). Turbidity was measured using portable turbidity meter (model HI 93 703–11). Temperature and dissolved oxygen (DO) were measured using a portable digital oxygen meter (EUTECH instruments, model DO300), pH was measured using digital pH meter (HANNA instruments, model HI 9024), while conductivity was determined using multi-parameter meter (HQ40d). The specific conductivity (K25) was calculated according to [31].
Analysis of physicochemical parameters
Total suspended solids (TSS) were determined gravimetrically using a known volume of properly mixed sample, which was filtered on pre-weighed glass fiber filter paper (GF/F) and pre-dried at 105 °C to constant weight to measure total suspended solids [32]. The titration method was used to estimate total alkalinity (TA) and phenolphthalein alkalinity (PA). Water samples were titrated with 0.2 N H2SO4 using phenolphthalein and bromocresol green-methyl red indicators within a few hours after sample collection according to [33] and expressed in meq L−1. Except for total phosphorus (TP) and ammonia (NH3 + NH4+-N), water samples were filtered with GF/F and used for spectrophotometric determination of inorganic nutrients. The ascorbic acid method was used to determine the soluble reactive phosphate- phosphorus (SRP) and total phosphorus (TP) after persulfate digestion [32]. The sodium salicylate method was used to determine nitrate (NO3-N), while the molybdosilicate method was used to estimate silica (SiO2) [32]. Finally, the phenate method was used to estimate ammonia [33].
Identification and enumeration of phytoplankton taxa
Samples were well mixed and 100 ml aliquots were transferred to 1000 ml measuring cylinders and kept in the dark for 24 h. Then, 90 ml of the supernatant was carefully drawn off. The remaining 10 ml was mixed properly and 1 ml was placed in a Sedgewick-Rafter cell and allowed to settle. Identification to genus/species level was done using various identification guides, e.g., [34, 35]. According to [36], cell abundance was estimated by counting cells of algal units encountered in 40–50 grids.
$${\text{Cells}}\;{\text{ml}}^{ - 1} = \frac{{N*1000\;{\text{mm}}^{3} }}{A*D*F}$$
where N is the number of cells or units counted, A is area of field (area of each grid, 1mm2), D is depth of Sedgwick-Rafter chamber (1 mm), and F is number of fields/grids counted.
Estimation of phytoplankton biomass
Phytoplankton biomass was estimated as chlorophyll-a (Chl-a) concentration (μg L−1) determined using a phytoplankton analyzer (PHYTO-PAM, Heinz Walz GmbH, Effeltrich, Germany).
Identification and quantification of cyanotoxins
Water samples were filtered using 47 mm GF/F filters. The seston retained by the filters was covered with aluminum foil and placed in Petri dishes and then kept in a freezer until analysis was conducted. The filtered water samples were kept in a freezer over night before analyses were conducted.
Extraction of MCs and NOD from algal seston
Extraction of MCs and NOD was done according to [37]. First, known volume of sample was filtered through a glass microfiber filter papers (47 mm GF/F filters). Then, the filter papers that retained algal seston were folded in half and put in a 10-ml glass tube and freeze-dried on a freeze-drier (Alpha 1–2 LD, Martin Christ Gefriertrocknung sanlagen GmbH, Osterode am Harz, Germany) for two hrs. After that, 2.5 ml of 75% methanol-25% Millipore water (v/v) was used to extract sample three times in a water bath at 60 °C using the glass pipette squeeze method. Then, 7.5 ml samples on the speedvac tube were dried up using a Speedvac (Savant SPD121P, Thermo Scientific, Waltham, MA, USA) centrifuge pre-heat at 50 °C. Then, 300 μl of 100% MeOH was used to reconstitute the sample three times after well vortexed and mixed. Afterward, the reconstituted 900μL samples were transferred to 2 ml Eppendorf vials with a cellulose-acetate filter (0.2 μm, Grace Davison Discovery Sciences, Deerfield, IL, USA) and centrifuged for 5 min at 16,000 × g (Galaxy 16DH, VWR, International). Finally, filtrates were transferred to amber glass vials and caps were tightly closed and stored in a freezer till LC–MS/MS analysis. Prior to LC–MS/MS analysis, calibration standards were prepared [38].
Extraction of extracellular microcystins and nodularin
Filtrates of water samples filtered using 47 mm GF/F filters and stored in a freezer were dried in a Speedvac (Savant SPD121P, pre-heat centrifuge at 50 °C). Then, 300 μl of 100% MeOH was used to reconstitute the sample three times after well vortexed and mixed. Afterward, the reconstituted 900μL samples were transferred to 2-ml Eppendorf vials with a cellulose-acetate filter (0.2 μm, Grace Davison Discovery Sciences, Deerfield, IL, USA) and centrifuged for 5 min at 16,000 × g (Galaxy 16DH, VWR, International). Finally, filtrates were transferred to amber glass vials and caps were tightly closed and stored in a freezer till LC–MS/MS analysis. Prior to LC–MS/MS analysis, calibration standards were prepared [38].
LC–MS/MS analyses
The concentrations of nine MC variants (dm-7-MC-RR, MC-RR, MC-YR, dm-7-MC-LR, MC-LR, MC-LA, MC-LY, MC-LW, and MC-LF) and nodularin (NOD) were examined by LC–MS/MS according to the procedure of [39]. LC–MS/MS analyses were done using an Agilent 1200 LC and an Agilent 6410A QQQ (Agilent Technologies, Santa Clara, CA, USA). MCs variants were separated on an Agilent Eclipse XDB-C18 4.6_150 mm, 5 m columns. Calibration standards were obtained from DHI LAB Products (Hørsholm, Denmark) [37] (supplementary material). The sample was injected with a flow rate of 0.5 ml/min; the column temperature was 40 °C. Eluents were Millipore water with 0.1% formic acid (v/v, Eluent A) and acetonitrile with 0.1% formic acid (v/v, Eluent B) that were run using an elution program of 0–2 min 30% B, 6–12 min 90% B, with a linear increase of B between 2 and 6 min and a 5-min post run at 30% B. Detailed information on MS/MS settings for each MC variant and NOD are shown in (supplementary material). Recovery of sample and analysis was estimated by spiking a cyanobacterial matrix in triplicates: The recovery rate was 100% for MC-LA, 99.5% for DM-7-MC-RR, 96.1% for MC-RR, 75% for MC-YR, 77.8% for DM-7-MC-RR, 79.1 for MC-LR, 72.8% for MC-LY, 53.5% for MC-LW, and 63.9% for MC-LF. Furthermore, repeatability, the limit of detection and limit of quantification of LC–MS/MS analyses are described in [38].