A report of Cylindrospermopsis raciborskii and other cyanobacteria in the water reservoirs of power plants in Ukraine

A report of Cylindrospermopsis raciborskii and other cyanobacteria in the water reservoirs of... The occurrence of cyanobacteria in freshwaters attracts much attention due to its associated health threats and ecological implications. Yet data on the composition of cyanobacteria taxa and toxigenicity in some regions is still scarce. Here, we explored the occurrence of cyanobacteria and cyanotoxins in three locations in Ukraine (reservoir for Kasperivtsi Hydrothermal Power Plant and outflowing River Seret, and cooling pond of Khmelnytsky Atomic Power Plant) in summer 2017. Cyanobacteria were a dominant fraction at all stations. A number of potent-toxin producers were identified including Cylindrospermopsis raciborskii, Aphanizomenon gracile, Dolichospermum flos-aquae,and Planktothrix agardhii. Screening for the presence of dissolved and particulate content of microcystins (-LR, -YR, and -RR), cylindrospermopsin, and anatoxin-a yielded negative results. The studied waters displayed no toxicity in human platelets in vitro. Further toxicological and ecological studies are necessary to evaluate the potential presence of cyanotoxin producers in Ukraine. . . . Keywords Cylindrospermopsis raciborskii Ukraine Cyanotoxins Cyanobacteria Introduction their nearly global distribution, potent production of various toxins, wide range of ecological adaptations, and ability to Cyanobacteria from the Chroococcales, Nostocales, and expand to new habitats. Their occurrence in freshwaters is Oscillatoriales orders are subject to ongoing interest due to promoted by cultural eutrophication but can also be driven by climate changes. As predicted, the incidence of harmful cyanobacteria blooms in various regions will increase advo- cating a need to monitor toxigenic species in various regions Responsible editor: Vitor Manuel Oliveira Vasconcelos (Paerl and Paul 2011;O’Neil et al. 2012). Particular attention is being paid to species that are recog- * Piotr Rzymski nized as potent producers of cyclic hepatopeptides rzymskipiotr@ump.edu.pl microcystins (MCs) that include Microcystis aeruginosa (Kütz.) Kützing, Planktothrix agardhii (Gomont) Department of Environmental Medicine, Poznan University of Anagnostidis & Komárek, and members of Nostoc, Medical Sciences, Poznań,Poland Anabaena/Dolichospermum,and Anabaenopsis (Bernard Research Laboratory of Comparative Biochemistry and Molecular et al. 2017), as well as potent producers of the neurotoxic Biology, Ternopil National Pedagogical University, (homo)anatoxin-a (ANA-A) alkaloid that encompass species Ternopil, Ukraine belonging to the Anabaena, Aphanizomenon,and Department of Water Protection, Adam Mickiewicz University, Dolichospermum genera. Moreover, the occurrence of cyto- Poznań,Poland toxic cylindrospermopsin (CYN) in European freshwater has Department of Applied Ecology, Faculty of Biology and been increasingly reported with strains of Aphanizomenon Environmental Protection, University of Łódź, Łódź, Poland gracile (Lemm.) Lemmermann, A. klebahnii Elenkin ex Department of Hydrobiology, Adam Mickiewicz University, Pechar, A. flos-aquae Ralfs ex Bornet et Flahault, A. Poznań,Poland ovalisporum Forti, and Oscillatoria sp. recognized as potent Department of Analytical Chemistry, Adam Mickiewicz University, producers (Rzymski and Poniedziałek 2014). Poznań,Poland 15246 Environ Sci Pollut Res (2018) 25:15245–15252 In turn, Cylindrospermopsis raciborskii (Woloszyńska) Phytoplankton analyses Seenayya et Subba Raju, known as the main producer of CYN in sub-tropical and tropical areas, has never been docu- The samples were preserved with Lugol solution and then mented to produce any known cyanotoxin in Europe although analyzed within a month from sampling. Qualitative and numerous studies have already demonstrated the toxicity of quantitative phytoplankton analysis was performed in a their exudates using in vivo and in vitro experimental models Sedgwick-Rafter chamber of 0.5 mL volume, with the use of (Acs et al. 2013; Smutná et al. 2016; Rzymski et al. 2017a). a light microscope Olympus BX60, according to standard Reported first in Lake Kastoria in Greece (Skuja 1937), it has methods (Wetzel and Likens 1991). Cyanobacterial and eu- systematically been found throughout the continent, although karyotic phytoplankton species were identified on the basis of was rarely observed to form blooms (Budzyńska and Gołdyn the microscopic analysis of their morphological features, ac- 2017). With recent reports of its common occurrence in west- cording to modern keys based on the polyphasic taxonomical ern parts of Poland, in a single lake in Lithuania (Kokocinski approach (Komárek and Anagnostidis 1998; Komárek and et al. 2017; Rzymski et al. 2017b) and Lake Nero in the Anagnostidis 2005;Komárek 2013). Yaroslavl Region of Russia (Babanazarova et al. 2015), it appears that C. raciborskii continues to spread. Its distribution Physicochemical water analyses pattern and threats associated with its expansion in Eastern Europe are, however, still not sufficiently explored. Water parameters were measured by routine analytical tests. Here, we report the occurrence of C. raciborskii and other pH was measured using an MI 150 tester with a combination cyanobacteria belonging to the Chroococcales, Nostocales, electrode. In the field, water for chemical analyses was placed and Oscillatoriales orders in three locations in Ukraine (water without preservation into polyethylene flasks and transported reservoir for Kasperivtsi Hydrothermal Power Plant and in coolers to the laboratory. The following water parameters outflowing River Seret and cooling pond of Khmelnytsky were analyzed: nitrogen forms (N-NH using the colorimet- Atomic Power Plant) along with analyses of cyanotoxins ric method with Nessler reagent, N-NO colorimetric with (CYN, MCs, and ANA-A), physicochemical parameters, Griess reagent, N-NO colorimetric with sodium salicylate) and in vitro toxicity of water. and total reactive phosphorus (TRP, using the molybdate method) (APHA 1995). Phenol concentration was evaluated using the 4-aminoantipyrine method (Dannis 1951). The chlo- Material and methods ride ion concentration was analyzed using the trimetric with silver nitrate method (EPA norm 9253 1994). Sulfate concen- Study area and water sampling tration in water samples was determined by indirect EDTA titration (Belle-Oudry 2008). The samplings were carried out during August and September Concentrations of calcium (Ca), magnesium (Mg), sodium 2017 according to Environmental Protection Agency recom- (Na), potassium (K), manganese (Mn), iron (Fe), aluminum mendations (Surface Water Sampling 2013). Surface water (Al), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), samples were collected at three sites (KHPP, RS, and KAPP) mercury (Hg), nickel (Ni), lead (Pb), and zinc (Zn) were quan- located in the Galicia-Volyn area, Western Ukraine (Fig. 1), tified with the inductively coupled plasma optical emission transferred to propylene bottles, and transported to the labora- spectrometer Agilent 5100 ICP-OES (Agilent, USA) after tory for determination of chemical parameters, phytoplankton acidification of samples with nitric acid (Sigma-Aldrich, analyses, and toxicological studies. The Kasperivtsi hydroelec- Germany) with conditions, wavelengths, and limit of detec- tric power plant is situated near the Seret riverbed (48° 40′ N, tion (LOD) as described previously (Rzymski et al. 2017c). 25° 51′ E). It is a small power plant with the installed capacity Calibration was performed using standard analytical solutions of 7.5 MW. Water discharged from the Kasperivtsi dammed (Merck, Germany). water reservoir (KHPP site) flows through a turbine directly into the River Seret (RS site). The Kasperivtsi water reservoir Cyanotoxin analyses (surface area 2.86 km and length 12 km) is a recreation area and the part of territory of National Nature Park BDnister Water samples in volume from 200 to 500 mL were filtered Canyon^ where no industrial contamination is expected. The through GF/C filters (Whatman, UK) to separate KAPP site, with a consistently higher water temperature, is cyanobacterial cells from water and to determine dissolved located on the bank of the cooling pond of Khmelnytsky and particulate concentration of cyanotoxins (microcystins Atomic Power Plant (APP) in Netishyn (in a forestry area on (MCs), anatoxin-a (ATX), and cylindrospermopsin (CYN)) the tributary of the River Goryn, 50° 21′ N, 26° 38′ E). There is using the HPLC-DAD method. no connection via water between these sites; the distance be- MCs in the suspended material were extracted in 75% tween the KHPP/RS and KAPP sites is about 300 km. aqueous methanol and CYN and ATX were extracted in Environ Sci Pollut Res (2018) 25:15245–15252 15247 Fig. 1 Localization of the sampling sites in Western Ukraine. Sites: A (KHPP)—Kasperivtsi water reservoir before dam; B (RS)—River Seret outflowing from Kasperivtsi water reservoir; C (KAPP)—cooling pond of Khmelnytsky Atomic Power Plant methanol. The samples were sonicated for 30 s in a Misonix cartridges (sorbent mass 200 mg) and C solid phase extrac- (Farmingdale, NY, USA) ultrasonicator equipped with an ul- tion cartridges were used in series (C before PGC). For trasonic probe (100 W, diameter 19 mm with Bspike^) and the conditioning of the combined system, 10 mL of methanol liquid processor XL. The extracts were then centrifuged twice containing 0.1% (v/v) TFA followed by 10 mL of water was at 11000×g for 10 min at 4 °C in an Eppendorf 5804 centri- used. CYN were eluted by 3 mL of 0.1% (v/v) TFA in meth- fuge (Hamburg, Germany). The supernatants were collected anol from PGC cartridges and then evaporated to dryness. and evaporated in a SC110A Speedvac Plus, ThermoSavant Before HPLC analysis, the samples were redissolved in (Holbrook, NY, USA). 75% aqueous methanol for MC analyses and in water for For dissolved MCs and ATX, samples of filtered water CYN and ATX analyses and filtered through a Gelman GHP were concentrated on Baker (Deventer, Netherlands) C solid Acrodisc 13-mm syringe filter with a 0.45-μmGHP membrane phase extraction (SPE) cartridges (sorbent mass 500 mg), con- and minispike outlet (East Hills, NY, USA). Chromatographic ditioned earlier by 10 mL of methanol and water. Cyanotoxins separation was performed using an Agilent (Waldbronn, were eluted from the C cartridges by 3 mL of 90% aqueous Germany) 1100 series HPLC system consisting of a degasser, methanol containing 0.1% trifluoroacetic acid (TFA). The el- a quaternary pump, a column compartment thermostat set at uates were evaporated to dryness in a SC110A Speedvac 40 °C, and a diode array detector operated at 200–300 nm on a Plus, ThermoSavant (Holbrook, NY, USA). For dissolved Merck (Darmstadt, Germany) Purospher STAR RP-18e col- CYN polygraphite carbon (PGC), solid phase extraction umn (55 mm × 4 mm I.D. with 3-μm particles) protected by a 15248 Environ Sci Pollut Res (2018) 25:15245–15252 4 mm × 4 mm guard column. The mobile phase consisted of buffer to evaluate the maximum releasable LDH activity water (solvent A) and acetonitrile (solvent B), both containing for each sample. 0.05% trifluoroacetic acid. The flow rate was 1.0 mL/min with the following linear gradient program: 0 min, 1% B; 5 min, 7% B; 5.1 min, 70% B; 7 min, 70% B; 7.1 min, 1% B; stop time, Results and discussion 12 min for CYN and ATX analyses. The injection volume was 20 μL. CYN in the samples was identified by comparing the The physicochemical parameters of water during the sampling retention time and UV spectrum (200–300 nm) with an absorp- period are summarized in Table 1. The concentrations of toxic tion maximum at 262 nm for CYN and at 227 nm for ATX. The elements were low, indicating no industrial pollution although −1 flow rate for MC analyses was 1.0 mL min with the following one should note that metal levels in water can be a subject to linear gradient program: 0 min, 25% B; 5 min, 70% B; 6 min, seasonal variations (Rzymski et al. 2014a). High sulfate con- 70% B; 6.1 min, 25% B; stop time, 9 min. The injection vol- centrations, exceeding threefold the maximum allowance lev- ume was 20 μL. The contents of MC-LR, MC-YR, and MC- el set for drinking water in the European Union (Directive 98/ RR in the samples were analyzed by comparing the retention 83/EC 1998), were noted at all sampling stations in August. time and UV spectrum (200–300 nm with an absorption max- Periodically increased levels of this parameter are observed in imum at 238 nm). surface waters of this region and can be, at least partially, explained by high sulfate concentration in soil (Choban and In vitro toxicity assessment Winkler 2010; Peryt et al. 2012). In general, the studied waters were eutrophic as indicated by inorganic nitrogen and phos- The cytotoxicity of sampled water was assessed in an in vitro phate concentrations. This was also evident from phytoplank- experimental model employing platelet-rich plasma (PRP) ton analyses—cyanobacteria were a dominant fraction in all and assessing lactate dehydrogenase (LDH) leakage. This the studied samples in both August and September, with abun- model was selected as cyanobacterial compounds can exhibit dance ranging from 78% to as much as 98% depending on toxicity in platelets (Selheim et al. 2005). PRP was isolated by period and studied site (Fig. 2). A share of each identified centrifugation (200×g, 12 min) from blood collected from five cyanobacterial species in the total phytoplankton and healthy donors at the Regional Centre of Blood and Blood cyanobacteria community is given in Table 2. The dominant Treatment in Poznan, Poland, according to accepted safeguard taxa at KHPP and RS sites included Pseudanabaena sp. and standards and legal requirements in Poland. One milliliter of Planktothrix agardhii (Fig. 3c), whereas P. agardhii and PRP was incubated with 100 μL of filtered water samples for Planktolyngbya limnetica dominated at the KAPP site. M. 1 h at 37 °C in darkness. Negative and positive controls were aeruginosa was identified only at the KAPP site and only in constituted of PRP incubated with phosphate-buffered saline September with a 17% share in total phytoplankton. C. and 10 μM of tert-butyl hydroperoxide (tBHP), respectively. raciborskii (Fig. 3a) and Sphaerospermopsis The cytotoxicity was evaluated using Cytotoxicity Detection aphanizomenoides (Fig. 3d) were identified at the KHPP LDH Kit (Sigma-Aldrich, Germany) according to the manu- and RS sites with their share in total phytoplankton reaching facturer’s instructions. Briefly, following the incubation, all a maximum of 8.5 and 6.3%, respectively. A. gracile was samples were centrifuged for 10 min at 1000 rpm and super- identified at all studied sites but at very low abundances natants (100 μL) were transferred to a 96-well flat bottom (Table 2). microplate and mixed with a 100-μL reaction cocktail con- This report highlights the presence of number of po- taining iodonitrotetrazolium chloride, sodium lactate, and di- tent toxin-producing cyanobacteria in man-made water aphorase/NAD+ mixture. After incubation (30 min, 25 °C, reservoirs in Ukraine. Although species such as C. darkness), the absorbance of each sample was read at raciborskii and M. aeruginosa have been reported earlier 492 nm, and the cytotoxicity of the lake water sample was in Ukraine (Novoselova and Protasov 2016), the data on calculated according to the equation: their potential toxicity is scarce and focused only on MCs. As provided by Belykh et al. (2013), screening of mcy genes in the freshwater of Kiev regions yielded Cytotoxicity½ % positive results in the majority of studied samples, al- though exact producers were not identified. Previous AbsorbanceðÞ sample −AbsorbanceðÞ control phytoplankton screening conducted in 2005 in the AbsorbanceðÞ high control −AbsorbanceðÞ control Kasperivtsi Hydrothermal Power Plant reservoir and outflowing River Seret did not identify C. raciborskii, P. agardhii,or A. gracile (Shcherbak and Bondarenko where control is a non-exposed sample and high control 2005). Therefore, the findings of the present study indi- represents a non-exposed sample mixed with RIPA lysis cate the potential expansion of these cyanobacteria in Environ Sci Pollut Res (2018) 25:15245–15252 15249 Table 1 Physicochemical parameters of water at sampling sites Parameter KHPP RS KAPP August September August September August September Temperature [°C] 23.3 15.1 25.2 14.9 28.1 17.7 рН 7.45 ± 0.06 8.00 ± 0.05 7.52 ± 0.07 7.77 ± 0.05 8.10 ± 0.05 7.80 ± 0.05 NH [mg/L] 0.23 ± 0.03 0.85 ± 0.05 0.26 ± 0.03 1.25 ± 0.25 0.33 ± 0.02 1.55 ± 0.75 NO [mg/L] 0.01 ± 0.005 0.11 ± 0.01 0.01 ± 0.005 0.11 ± 0.005 0.01 ± 0.005 0.01 ± 0.01 NO [mg/L] 0.49 ± 0.05 1.45 ± 0.19 0.53 ± 0.06 2.10 ± 0.09 0.20 ± 0.02 0.03 ± 0.05 Cl [mg/L] 40.32 ± 1.50 28.36 ± 1.75 39.10 ± 1.50 29.00 ± 1.65 49.63 ± 2.15 56.72 ± 2.50 2− SO [mg/L] 796.0 ± 76.2 220.0 ± 15.6 740.0 ± 57.7 240.0 ± 20.0 744.5 ± 33.9 150.5 ± 12.9 2+ PO [mg/L] 0.41 ± 0.005 0.14 ± 0.02 0.38 ± 0.005 0.13 ± 0.03 0.386 ± 0.002 0.16 ± 0.007 Phenol [μg/L] 0.20 ± 0.02 0.23 ± 0.02 0.18 ± 0.02 0.22 ± 0.05 1.00 ± 0.04 0.13 ± 0.04 Dry residue [mg/L] 748 ± 31 825 702 ± 35 750 916 ± 43 725 Total Ca [mg/L] 61.2 ± 1.9 76.1 ± 1.2 65.7 ± 1.3 78.4 ± 1.8 68.3 ± 2.6 65.9 ± 1.4 Total Fe [mg/L] 0.07 ± 0.01 0.04 ± 0.005 0.25 ± 0.1 0.03 ± 0.002 0.10 ± 0.001 0.02 ± 0.001 Total K [mg/L] 5.52 ± 0.6 5.88 ± 0.2 4.70 ± 0.1 5.19 ± 0.3 7.26 ± 0.3 6.42 ± 0.2 Total Mg [mg/L] 9.25 ± 0.5 8.24 ± 0.1 8.6 ± 0.1 8.53 ± 0.1 8.52 ± 0.3 8.1 ± 0.2 Total Na [mg/L] 10.51 ± 0.3 10.2 ± 0.1 10.28 ± 0.1 10.0 ± 0.2 39.52 ± 1.5 36.8 ± 0.2 Total Mn [mg/L] 0.06 ± 0.3 0.05 ± 0.002 0.18 ± 0.08 0.04 ± 0.01 0.05 ± 0.03 0.01 ± 0.01 Total Al [mg/L] 0.55 ± 0.2 0.15 ± 0.06 0.044 ± 0.03 0.019 ± 0.01 0.01 ± 0.001 0.014 ± 0.001 Total Cd [mg/L] < LOD 0.002 ± 0.001 0.001 ± 0.001 < LOD < LOD < LOD Total Co [mg/L] 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.005 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 Total Cr [mg/L] 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.005 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 Total Cu [mg/L] < LOD 0.005 ± 0.001 < LOD 0.015 ± 0.001 0.005 ± 0.001 0.032 ± 0.001 Total Hg [mg/L] < LOD < LOD < LOD < LOD < LOD < LOD Total Ni [mg/L] 0.007 ± 0.001 0.007 ± 0.001 0.004 ± 0.001 < LOD 0.012 ± 0.001 0.008 ± 0.001 Total Pb [mg/L] 0.011 0.018 0.045 0.075 0.024 0.042 Total Zn [mg/L] < LOD < LOD < LOD < LOD < LOD 0.005 ± 0.001 <LOD below limit of detection this region and highlight that their further dispersion content of all three studied MC analogues was found below through a river system is plausible. detection limits. However, the occurrence of non-MC- Our study identified two main potent producers of MCs: M. producing strains of both species is a common phenomenon aeruginosa and P. agardhii, but even though the latter reaches (Yéprémian et al. 2007;Park etal. 2018), and previous studies a relatively high abundance, the dissolved and particulate have indicated that the temporal and spatial distribution of Fig. 2 The abundance of cyanobacteria at three studied sites in August and September 2017 15250 Environ Sci Pollut Res (2018) 25:15245–15252 Table 2 The cyanobacteria species identified at studied sites Share in total phytoplankton (%) Share in total cyanobacteria (%) August/September KHPP RS KAPP KHPP RS KAPP Anabaenopsis cunningtonnii 0.0/0.1 0.3/0.8 0.0/0.0 0.0/0.2 0.3/0.9 0.0/0.0 Anabaenopsis elenkinii 0.0/0.0 0.1/0.0 0.0/0.0 0.0/0.0 0.1/0.0 0.0/0.0 Aphanizomenon gracile 1.5/6.9 1.5/3.9 0.3/0.0 1.5/7.4 1.6/4.2 0.4/0.0 Cuspidothrix issatschenkoi 0.3/0.0 0.3/0.0 0.0/0.0 0.3/0.0 0.3/0.0 0.0/0.0 Cylindrospermopsis raciborskii 3.9/8.5 2.7/1.1 0.0/0.0 4.0/9.2 2.8/1.1 0.0/0.0 Dolichospermum sp. 0.3/2.3 0.0/2.4 0.0/0.0 0.3/2.5 0.0/2.5 0.0/0.0 Dolichospermum flos-aquae 1.3/0.5 0.7/7.1 0.0/0.0 1.4/0.6 0.7/7.5 0.0/0.0 Limnothrix redeckei 0.7/0.0 0.1/0.3 0.0/0.0 0.7/0.0 0.7/0.3 0.0/0.0 Merismopedia tenuissima 0.1/0.0 0.0/0.6 0.0/0.0 0.2/0.0 0.0/0.6 0.0/0.0 Microcystis aeruginosa 0.0/0.0 0.4/0.0 0.0/17.0 0.0/0.0 0.4/0.0 0.0/18.4 Planktothrix agardhii 25.9/38.8 72.3/34.1 54.7/75.5 26.8/42.0 73.8/36.3 69.6/81.6 Planktolyngbya limnetica 8.2/0.0 1.4/0.0 18.2/0.0 8.4/0.0 1.4/0.0 23.1/0.0 Pseudanabaena sp. 49.0/35.3 12.0/43.8 5.4/0.0 50.6/38.2 12.2/46.6 6.8/0.0 Sphaerospermopsis aphanizomenoides 5.6/0.0 6.2/0.0 0.0/0.0 5.8/0.0 6.3/0.0 0.0/0.0 toxic and non-toxic genotypes can be attributed to a number of identified strain of A. gracile (Fig. 1b), a species previously factors including resource competition (Briand et al. 2008;Lei demonstrated as a major CYN producer in Poland et al. 2015;Suominen etal. 2017). (Kokociński et al. 2013). Our findings also support a prelim- Potential ANA-A-producing cyanobacteria identified in inary view that Ukrainian strains of C. raciborskii are unable this study included Cuspidothrix issatschenkoi (Usačev) to produce CYN. The abundance of this species in the studied Rajaniemi et al. (Ballot et al. 2010) and Dolichospermum waters was relatively low, in line with previous observations flos-aquae (Lyngbye) Wacklin, Hoffmann et Komárek in Polish lakes where C. raciborskii biomass, even during (Osswald et al. 2009), although this neurotoxin was not de- cyanobacterial blooms, was usually not high, and maximally tected. Moreover, no dissolved or particulate CYN was found accounted for 40% of total phytoplankton biomass in the studied samples, excluding its production by the (Kokocinski et al. 2017). However, there was a single reported Fig. 3 Cyanobacteria identified at KHPP and RS sites. a Cylindrospermopsis raciborskii, b Aphanizomenon gracile, c Planktothrix agardhii,and d Sphaerospermopsis aphanizomenoides Environ Sci Pollut Res (2018) 25:15245–15252 15251 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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Springer Verlag, 163–173 New York, Berlin, Heidelberg, pp. 391 Peryt TM, Durakiewicz T, Peryt D, Poberezhskyy A (2012) Yéprémian C, Gugger MF, Briand E, Catherine A, Berger C, Quiblier C, Carbon and oxygen isotopic composition of the Middle Bernard C (2007) Microcystin ecotypes in a perennial Planktothrix Miocene. Geol Acta 10:1–12 agardhii bloom. Water Res 41:4446–4456 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Science and Pollution Research Springer Journals

A report of Cylindrospermopsis raciborskii and other cyanobacteria in the water reservoirs of power plants in Ukraine

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Environment; Environment, general; Environmental Chemistry; Ecotoxicology; Environmental Health; Atmospheric Protection/Air Quality Control/Air Pollution; Waste Water Technology / Water Pollution Control / Water Management / Aquatic Pollution
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0944-1344
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10.1007/s11356-018-2010-6
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Abstract

The occurrence of cyanobacteria in freshwaters attracts much attention due to its associated health threats and ecological implications. Yet data on the composition of cyanobacteria taxa and toxigenicity in some regions is still scarce. Here, we explored the occurrence of cyanobacteria and cyanotoxins in three locations in Ukraine (reservoir for Kasperivtsi Hydrothermal Power Plant and outflowing River Seret, and cooling pond of Khmelnytsky Atomic Power Plant) in summer 2017. Cyanobacteria were a dominant fraction at all stations. A number of potent-toxin producers were identified including Cylindrospermopsis raciborskii, Aphanizomenon gracile, Dolichospermum flos-aquae,and Planktothrix agardhii. Screening for the presence of dissolved and particulate content of microcystins (-LR, -YR, and -RR), cylindrospermopsin, and anatoxin-a yielded negative results. The studied waters displayed no toxicity in human platelets in vitro. Further toxicological and ecological studies are necessary to evaluate the potential presence of cyanotoxin producers in Ukraine. . . . Keywords Cylindrospermopsis raciborskii Ukraine Cyanotoxins Cyanobacteria Introduction their nearly global distribution, potent production of various toxins, wide range of ecological adaptations, and ability to Cyanobacteria from the Chroococcales, Nostocales, and expand to new habitats. Their occurrence in freshwaters is Oscillatoriales orders are subject to ongoing interest due to promoted by cultural eutrophication but can also be driven by climate changes. As predicted, the incidence of harmful cyanobacteria blooms in various regions will increase advo- cating a need to monitor toxigenic species in various regions Responsible editor: Vitor Manuel Oliveira Vasconcelos (Paerl and Paul 2011;O’Neil et al. 2012). Particular attention is being paid to species that are recog- * Piotr Rzymski nized as potent producers of cyclic hepatopeptides rzymskipiotr@ump.edu.pl microcystins (MCs) that include Microcystis aeruginosa (Kütz.) Kützing, Planktothrix agardhii (Gomont) Department of Environmental Medicine, Poznan University of Anagnostidis & Komárek, and members of Nostoc, Medical Sciences, Poznań,Poland Anabaena/Dolichospermum,and Anabaenopsis (Bernard Research Laboratory of Comparative Biochemistry and Molecular et al. 2017), as well as potent producers of the neurotoxic Biology, Ternopil National Pedagogical University, (homo)anatoxin-a (ANA-A) alkaloid that encompass species Ternopil, Ukraine belonging to the Anabaena, Aphanizomenon,and Department of Water Protection, Adam Mickiewicz University, Dolichospermum genera. Moreover, the occurrence of cyto- Poznań,Poland toxic cylindrospermopsin (CYN) in European freshwater has Department of Applied Ecology, Faculty of Biology and been increasingly reported with strains of Aphanizomenon Environmental Protection, University of Łódź, Łódź, Poland gracile (Lemm.) Lemmermann, A. klebahnii Elenkin ex Department of Hydrobiology, Adam Mickiewicz University, Pechar, A. flos-aquae Ralfs ex Bornet et Flahault, A. Poznań,Poland ovalisporum Forti, and Oscillatoria sp. recognized as potent Department of Analytical Chemistry, Adam Mickiewicz University, producers (Rzymski and Poniedziałek 2014). Poznań,Poland 15246 Environ Sci Pollut Res (2018) 25:15245–15252 In turn, Cylindrospermopsis raciborskii (Woloszyńska) Phytoplankton analyses Seenayya et Subba Raju, known as the main producer of CYN in sub-tropical and tropical areas, has never been docu- The samples were preserved with Lugol solution and then mented to produce any known cyanotoxin in Europe although analyzed within a month from sampling. Qualitative and numerous studies have already demonstrated the toxicity of quantitative phytoplankton analysis was performed in a their exudates using in vivo and in vitro experimental models Sedgwick-Rafter chamber of 0.5 mL volume, with the use of (Acs et al. 2013; Smutná et al. 2016; Rzymski et al. 2017a). a light microscope Olympus BX60, according to standard Reported first in Lake Kastoria in Greece (Skuja 1937), it has methods (Wetzel and Likens 1991). Cyanobacterial and eu- systematically been found throughout the continent, although karyotic phytoplankton species were identified on the basis of was rarely observed to form blooms (Budzyńska and Gołdyn the microscopic analysis of their morphological features, ac- 2017). With recent reports of its common occurrence in west- cording to modern keys based on the polyphasic taxonomical ern parts of Poland, in a single lake in Lithuania (Kokocinski approach (Komárek and Anagnostidis 1998; Komárek and et al. 2017; Rzymski et al. 2017b) and Lake Nero in the Anagnostidis 2005;Komárek 2013). Yaroslavl Region of Russia (Babanazarova et al. 2015), it appears that C. raciborskii continues to spread. Its distribution Physicochemical water analyses pattern and threats associated with its expansion in Eastern Europe are, however, still not sufficiently explored. Water parameters were measured by routine analytical tests. Here, we report the occurrence of C. raciborskii and other pH was measured using an MI 150 tester with a combination cyanobacteria belonging to the Chroococcales, Nostocales, electrode. In the field, water for chemical analyses was placed and Oscillatoriales orders in three locations in Ukraine (water without preservation into polyethylene flasks and transported reservoir for Kasperivtsi Hydrothermal Power Plant and in coolers to the laboratory. The following water parameters outflowing River Seret and cooling pond of Khmelnytsky were analyzed: nitrogen forms (N-NH using the colorimet- Atomic Power Plant) along with analyses of cyanotoxins ric method with Nessler reagent, N-NO colorimetric with (CYN, MCs, and ANA-A), physicochemical parameters, Griess reagent, N-NO colorimetric with sodium salicylate) and in vitro toxicity of water. and total reactive phosphorus (TRP, using the molybdate method) (APHA 1995). Phenol concentration was evaluated using the 4-aminoantipyrine method (Dannis 1951). The chlo- Material and methods ride ion concentration was analyzed using the trimetric with silver nitrate method (EPA norm 9253 1994). Sulfate concen- Study area and water sampling tration in water samples was determined by indirect EDTA titration (Belle-Oudry 2008). The samplings were carried out during August and September Concentrations of calcium (Ca), magnesium (Mg), sodium 2017 according to Environmental Protection Agency recom- (Na), potassium (K), manganese (Mn), iron (Fe), aluminum mendations (Surface Water Sampling 2013). Surface water (Al), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), samples were collected at three sites (KHPP, RS, and KAPP) mercury (Hg), nickel (Ni), lead (Pb), and zinc (Zn) were quan- located in the Galicia-Volyn area, Western Ukraine (Fig. 1), tified with the inductively coupled plasma optical emission transferred to propylene bottles, and transported to the labora- spectrometer Agilent 5100 ICP-OES (Agilent, USA) after tory for determination of chemical parameters, phytoplankton acidification of samples with nitric acid (Sigma-Aldrich, analyses, and toxicological studies. The Kasperivtsi hydroelec- Germany) with conditions, wavelengths, and limit of detec- tric power plant is situated near the Seret riverbed (48° 40′ N, tion (LOD) as described previously (Rzymski et al. 2017c). 25° 51′ E). It is a small power plant with the installed capacity Calibration was performed using standard analytical solutions of 7.5 MW. Water discharged from the Kasperivtsi dammed (Merck, Germany). water reservoir (KHPP site) flows through a turbine directly into the River Seret (RS site). The Kasperivtsi water reservoir Cyanotoxin analyses (surface area 2.86 km and length 12 km) is a recreation area and the part of territory of National Nature Park BDnister Water samples in volume from 200 to 500 mL were filtered Canyon^ where no industrial contamination is expected. The through GF/C filters (Whatman, UK) to separate KAPP site, with a consistently higher water temperature, is cyanobacterial cells from water and to determine dissolved located on the bank of the cooling pond of Khmelnytsky and particulate concentration of cyanotoxins (microcystins Atomic Power Plant (APP) in Netishyn (in a forestry area on (MCs), anatoxin-a (ATX), and cylindrospermopsin (CYN)) the tributary of the River Goryn, 50° 21′ N, 26° 38′ E). There is using the HPLC-DAD method. no connection via water between these sites; the distance be- MCs in the suspended material were extracted in 75% tween the KHPP/RS and KAPP sites is about 300 km. aqueous methanol and CYN and ATX were extracted in Environ Sci Pollut Res (2018) 25:15245–15252 15247 Fig. 1 Localization of the sampling sites in Western Ukraine. Sites: A (KHPP)—Kasperivtsi water reservoir before dam; B (RS)—River Seret outflowing from Kasperivtsi water reservoir; C (KAPP)—cooling pond of Khmelnytsky Atomic Power Plant methanol. The samples were sonicated for 30 s in a Misonix cartridges (sorbent mass 200 mg) and C solid phase extrac- (Farmingdale, NY, USA) ultrasonicator equipped with an ul- tion cartridges were used in series (C before PGC). For trasonic probe (100 W, diameter 19 mm with Bspike^) and the conditioning of the combined system, 10 mL of methanol liquid processor XL. The extracts were then centrifuged twice containing 0.1% (v/v) TFA followed by 10 mL of water was at 11000×g for 10 min at 4 °C in an Eppendorf 5804 centri- used. CYN were eluted by 3 mL of 0.1% (v/v) TFA in meth- fuge (Hamburg, Germany). The supernatants were collected anol from PGC cartridges and then evaporated to dryness. and evaporated in a SC110A Speedvac Plus, ThermoSavant Before HPLC analysis, the samples were redissolved in (Holbrook, NY, USA). 75% aqueous methanol for MC analyses and in water for For dissolved MCs and ATX, samples of filtered water CYN and ATX analyses and filtered through a Gelman GHP were concentrated on Baker (Deventer, Netherlands) C solid Acrodisc 13-mm syringe filter with a 0.45-μmGHP membrane phase extraction (SPE) cartridges (sorbent mass 500 mg), con- and minispike outlet (East Hills, NY, USA). Chromatographic ditioned earlier by 10 mL of methanol and water. Cyanotoxins separation was performed using an Agilent (Waldbronn, were eluted from the C cartridges by 3 mL of 90% aqueous Germany) 1100 series HPLC system consisting of a degasser, methanol containing 0.1% trifluoroacetic acid (TFA). The el- a quaternary pump, a column compartment thermostat set at uates were evaporated to dryness in a SC110A Speedvac 40 °C, and a diode array detector operated at 200–300 nm on a Plus, ThermoSavant (Holbrook, NY, USA). For dissolved Merck (Darmstadt, Germany) Purospher STAR RP-18e col- CYN polygraphite carbon (PGC), solid phase extraction umn (55 mm × 4 mm I.D. with 3-μm particles) protected by a 15248 Environ Sci Pollut Res (2018) 25:15245–15252 4 mm × 4 mm guard column. The mobile phase consisted of buffer to evaluate the maximum releasable LDH activity water (solvent A) and acetonitrile (solvent B), both containing for each sample. 0.05% trifluoroacetic acid. The flow rate was 1.0 mL/min with the following linear gradient program: 0 min, 1% B; 5 min, 7% B; 5.1 min, 70% B; 7 min, 70% B; 7.1 min, 1% B; stop time, Results and discussion 12 min for CYN and ATX analyses. The injection volume was 20 μL. CYN in the samples was identified by comparing the The physicochemical parameters of water during the sampling retention time and UV spectrum (200–300 nm) with an absorp- period are summarized in Table 1. The concentrations of toxic tion maximum at 262 nm for CYN and at 227 nm for ATX. The elements were low, indicating no industrial pollution although −1 flow rate for MC analyses was 1.0 mL min with the following one should note that metal levels in water can be a subject to linear gradient program: 0 min, 25% B; 5 min, 70% B; 6 min, seasonal variations (Rzymski et al. 2014a). High sulfate con- 70% B; 6.1 min, 25% B; stop time, 9 min. The injection vol- centrations, exceeding threefold the maximum allowance lev- ume was 20 μL. The contents of MC-LR, MC-YR, and MC- el set for drinking water in the European Union (Directive 98/ RR in the samples were analyzed by comparing the retention 83/EC 1998), were noted at all sampling stations in August. time and UV spectrum (200–300 nm with an absorption max- Periodically increased levels of this parameter are observed in imum at 238 nm). surface waters of this region and can be, at least partially, explained by high sulfate concentration in soil (Choban and In vitro toxicity assessment Winkler 2010; Peryt et al. 2012). In general, the studied waters were eutrophic as indicated by inorganic nitrogen and phos- The cytotoxicity of sampled water was assessed in an in vitro phate concentrations. This was also evident from phytoplank- experimental model employing platelet-rich plasma (PRP) ton analyses—cyanobacteria were a dominant fraction in all and assessing lactate dehydrogenase (LDH) leakage. This the studied samples in both August and September, with abun- model was selected as cyanobacterial compounds can exhibit dance ranging from 78% to as much as 98% depending on toxicity in platelets (Selheim et al. 2005). PRP was isolated by period and studied site (Fig. 2). A share of each identified centrifugation (200×g, 12 min) from blood collected from five cyanobacterial species in the total phytoplankton and healthy donors at the Regional Centre of Blood and Blood cyanobacteria community is given in Table 2. The dominant Treatment in Poznan, Poland, according to accepted safeguard taxa at KHPP and RS sites included Pseudanabaena sp. and standards and legal requirements in Poland. One milliliter of Planktothrix agardhii (Fig. 3c), whereas P. agardhii and PRP was incubated with 100 μL of filtered water samples for Planktolyngbya limnetica dominated at the KAPP site. M. 1 h at 37 °C in darkness. Negative and positive controls were aeruginosa was identified only at the KAPP site and only in constituted of PRP incubated with phosphate-buffered saline September with a 17% share in total phytoplankton. C. and 10 μM of tert-butyl hydroperoxide (tBHP), respectively. raciborskii (Fig. 3a) and Sphaerospermopsis The cytotoxicity was evaluated using Cytotoxicity Detection aphanizomenoides (Fig. 3d) were identified at the KHPP LDH Kit (Sigma-Aldrich, Germany) according to the manu- and RS sites with their share in total phytoplankton reaching facturer’s instructions. Briefly, following the incubation, all a maximum of 8.5 and 6.3%, respectively. A. gracile was samples were centrifuged for 10 min at 1000 rpm and super- identified at all studied sites but at very low abundances natants (100 μL) were transferred to a 96-well flat bottom (Table 2). microplate and mixed with a 100-μL reaction cocktail con- This report highlights the presence of number of po- taining iodonitrotetrazolium chloride, sodium lactate, and di- tent toxin-producing cyanobacteria in man-made water aphorase/NAD+ mixture. After incubation (30 min, 25 °C, reservoirs in Ukraine. Although species such as C. darkness), the absorbance of each sample was read at raciborskii and M. aeruginosa have been reported earlier 492 nm, and the cytotoxicity of the lake water sample was in Ukraine (Novoselova and Protasov 2016), the data on calculated according to the equation: their potential toxicity is scarce and focused only on MCs. As provided by Belykh et al. (2013), screening of mcy genes in the freshwater of Kiev regions yielded Cytotoxicity½ % positive results in the majority of studied samples, al- though exact producers were not identified. Previous AbsorbanceðÞ sample −AbsorbanceðÞ control phytoplankton screening conducted in 2005 in the AbsorbanceðÞ high control −AbsorbanceðÞ control Kasperivtsi Hydrothermal Power Plant reservoir and outflowing River Seret did not identify C. raciborskii, P. agardhii,or A. gracile (Shcherbak and Bondarenko where control is a non-exposed sample and high control 2005). Therefore, the findings of the present study indi- represents a non-exposed sample mixed with RIPA lysis cate the potential expansion of these cyanobacteria in Environ Sci Pollut Res (2018) 25:15245–15252 15249 Table 1 Physicochemical parameters of water at sampling sites Parameter KHPP RS KAPP August September August September August September Temperature [°C] 23.3 15.1 25.2 14.9 28.1 17.7 рН 7.45 ± 0.06 8.00 ± 0.05 7.52 ± 0.07 7.77 ± 0.05 8.10 ± 0.05 7.80 ± 0.05 NH [mg/L] 0.23 ± 0.03 0.85 ± 0.05 0.26 ± 0.03 1.25 ± 0.25 0.33 ± 0.02 1.55 ± 0.75 NO [mg/L] 0.01 ± 0.005 0.11 ± 0.01 0.01 ± 0.005 0.11 ± 0.005 0.01 ± 0.005 0.01 ± 0.01 NO [mg/L] 0.49 ± 0.05 1.45 ± 0.19 0.53 ± 0.06 2.10 ± 0.09 0.20 ± 0.02 0.03 ± 0.05 Cl [mg/L] 40.32 ± 1.50 28.36 ± 1.75 39.10 ± 1.50 29.00 ± 1.65 49.63 ± 2.15 56.72 ± 2.50 2− SO [mg/L] 796.0 ± 76.2 220.0 ± 15.6 740.0 ± 57.7 240.0 ± 20.0 744.5 ± 33.9 150.5 ± 12.9 2+ PO [mg/L] 0.41 ± 0.005 0.14 ± 0.02 0.38 ± 0.005 0.13 ± 0.03 0.386 ± 0.002 0.16 ± 0.007 Phenol [μg/L] 0.20 ± 0.02 0.23 ± 0.02 0.18 ± 0.02 0.22 ± 0.05 1.00 ± 0.04 0.13 ± 0.04 Dry residue [mg/L] 748 ± 31 825 702 ± 35 750 916 ± 43 725 Total Ca [mg/L] 61.2 ± 1.9 76.1 ± 1.2 65.7 ± 1.3 78.4 ± 1.8 68.3 ± 2.6 65.9 ± 1.4 Total Fe [mg/L] 0.07 ± 0.01 0.04 ± 0.005 0.25 ± 0.1 0.03 ± 0.002 0.10 ± 0.001 0.02 ± 0.001 Total K [mg/L] 5.52 ± 0.6 5.88 ± 0.2 4.70 ± 0.1 5.19 ± 0.3 7.26 ± 0.3 6.42 ± 0.2 Total Mg [mg/L] 9.25 ± 0.5 8.24 ± 0.1 8.6 ± 0.1 8.53 ± 0.1 8.52 ± 0.3 8.1 ± 0.2 Total Na [mg/L] 10.51 ± 0.3 10.2 ± 0.1 10.28 ± 0.1 10.0 ± 0.2 39.52 ± 1.5 36.8 ± 0.2 Total Mn [mg/L] 0.06 ± 0.3 0.05 ± 0.002 0.18 ± 0.08 0.04 ± 0.01 0.05 ± 0.03 0.01 ± 0.01 Total Al [mg/L] 0.55 ± 0.2 0.15 ± 0.06 0.044 ± 0.03 0.019 ± 0.01 0.01 ± 0.001 0.014 ± 0.001 Total Cd [mg/L] < LOD 0.002 ± 0.001 0.001 ± 0.001 < LOD < LOD < LOD Total Co [mg/L] 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.005 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 Total Cr [mg/L] 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.005 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 Total Cu [mg/L] < LOD 0.005 ± 0.001 < LOD 0.015 ± 0.001 0.005 ± 0.001 0.032 ± 0.001 Total Hg [mg/L] < LOD < LOD < LOD < LOD < LOD < LOD Total Ni [mg/L] 0.007 ± 0.001 0.007 ± 0.001 0.004 ± 0.001 < LOD 0.012 ± 0.001 0.008 ± 0.001 Total Pb [mg/L] 0.011 0.018 0.045 0.075 0.024 0.042 Total Zn [mg/L] < LOD < LOD < LOD < LOD < LOD 0.005 ± 0.001 <LOD below limit of detection this region and highlight that their further dispersion content of all three studied MC analogues was found below through a river system is plausible. detection limits. However, the occurrence of non-MC- Our study identified two main potent producers of MCs: M. producing strains of both species is a common phenomenon aeruginosa and P. agardhii, but even though the latter reaches (Yéprémian et al. 2007;Park etal. 2018), and previous studies a relatively high abundance, the dissolved and particulate have indicated that the temporal and spatial distribution of Fig. 2 The abundance of cyanobacteria at three studied sites in August and September 2017 15250 Environ Sci Pollut Res (2018) 25:15245–15252 Table 2 The cyanobacteria species identified at studied sites Share in total phytoplankton (%) Share in total cyanobacteria (%) August/September KHPP RS KAPP KHPP RS KAPP Anabaenopsis cunningtonnii 0.0/0.1 0.3/0.8 0.0/0.0 0.0/0.2 0.3/0.9 0.0/0.0 Anabaenopsis elenkinii 0.0/0.0 0.1/0.0 0.0/0.0 0.0/0.0 0.1/0.0 0.0/0.0 Aphanizomenon gracile 1.5/6.9 1.5/3.9 0.3/0.0 1.5/7.4 1.6/4.2 0.4/0.0 Cuspidothrix issatschenkoi 0.3/0.0 0.3/0.0 0.0/0.0 0.3/0.0 0.3/0.0 0.0/0.0 Cylindrospermopsis raciborskii 3.9/8.5 2.7/1.1 0.0/0.0 4.0/9.2 2.8/1.1 0.0/0.0 Dolichospermum sp. 0.3/2.3 0.0/2.4 0.0/0.0 0.3/2.5 0.0/2.5 0.0/0.0 Dolichospermum flos-aquae 1.3/0.5 0.7/7.1 0.0/0.0 1.4/0.6 0.7/7.5 0.0/0.0 Limnothrix redeckei 0.7/0.0 0.1/0.3 0.0/0.0 0.7/0.0 0.7/0.3 0.0/0.0 Merismopedia tenuissima 0.1/0.0 0.0/0.6 0.0/0.0 0.2/0.0 0.0/0.6 0.0/0.0 Microcystis aeruginosa 0.0/0.0 0.4/0.0 0.0/17.0 0.0/0.0 0.4/0.0 0.0/18.4 Planktothrix agardhii 25.9/38.8 72.3/34.1 54.7/75.5 26.8/42.0 73.8/36.3 69.6/81.6 Planktolyngbya limnetica 8.2/0.0 1.4/0.0 18.2/0.0 8.4/0.0 1.4/0.0 23.1/0.0 Pseudanabaena sp. 49.0/35.3 12.0/43.8 5.4/0.0 50.6/38.2 12.2/46.6 6.8/0.0 Sphaerospermopsis aphanizomenoides 5.6/0.0 6.2/0.0 0.0/0.0 5.8/0.0 6.3/0.0 0.0/0.0 toxic and non-toxic genotypes can be attributed to a number of identified strain of A. gracile (Fig. 1b), a species previously factors including resource competition (Briand et al. 2008;Lei demonstrated as a major CYN producer in Poland et al. 2015;Suominen etal. 2017). (Kokociński et al. 2013). Our findings also support a prelim- Potential ANA-A-producing cyanobacteria identified in inary view that Ukrainian strains of C. raciborskii are unable this study included Cuspidothrix issatschenkoi (Usačev) to produce CYN. The abundance of this species in the studied Rajaniemi et al. (Ballot et al. 2010) and Dolichospermum waters was relatively low, in line with previous observations flos-aquae (Lyngbye) Wacklin, Hoffmann et Komárek in Polish lakes where C. raciborskii biomass, even during (Osswald et al. 2009), although this neurotoxin was not de- cyanobacterial blooms, was usually not high, and maximally tected. Moreover, no dissolved or particulate CYN was found accounted for 40% of total phytoplankton biomass in the studied samples, excluding its production by the (Kokocinski et al. 2017). However, there was a single reported Fig. 3 Cyanobacteria identified at KHPP and RS sites. a Cylindrospermopsis raciborskii, b Aphanizomenon gracile, c Planktothrix agardhii,and d Sphaerospermopsis aphanizomenoides Environ Sci Pollut Res (2018) 25:15245–15252 15251 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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Environmental Science and Pollution ResearchSpringer Journals

Published: Apr 21, 2018

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