Layout 1 INTRODUCTION Current estimates suggest that between 4000 and 5000 phytoplankton species have been described from inland waters (Reynolds, 1996, 2006). Among them are the prokaryotic phototrophs named Cyanoprokaryota (Cyanobacteria, Cyanophyta or Blue-green algae), which are also quantitatively amid the most important organisms on Earth (Whitton and Potts, 2012). Their appearance, traced back to the Early Archaean 3900 Ma ago (Graham et al., 2009), became a crucial step in the evolution of life in water and, subsequently, on land. The later origin and spread of their heterocytous forms, capable of nitrogen fixation, fits well with the timing of Great Oxidation Event about 2400 Ma ago (Schopf, 2012). Since then cyanoprokaryotes are the only nitrogen fixing organisms that also produce oxygen through photosynthesis, with in- creasing at steady pace number of known non-heterocy- tous species that possess this ability (Stal, 2012). Considering their additional attribute of buoyancy regu- lation through gas vacuoles, it is to understand why these peculiar organisms for years remained a fascinating topic (Reynolds, 2006) in biology, with increasing findings of their great potential as providers of ecosystem services. Cyanoprokaryotes are primary colonizers of various (even extreme) habitats, important basis of numerous food chains and have been repeatedly reported for their appli- cations in biotechnology, food industry and pharmacy (Whitton, 2012). However, during the last decades the recognition of the group turned in an almost universal contempt (Reynolds, 2006) due to their potential toxicity, key role in many harmful blooms and general assuming Advances in Oceanography and Limnology, 2017; 8(1): 131-152 ARTICLE DOI: 10.4081/aiol.2017.6320 This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). Assessment of cyanoprokaryote blooms and of cyanotoxins in Bulgaria in a 15-years period (2000-2015) Maya P. Stoyneva-Gärtner,1* Jean-Pierre Descy,2,3 Adrien Latli,3 Blagoy A. Uzunov,1 Vera T. Pavlova,4 Zlatka Bratanova,4 Pavel Babica,5,6 Blahoslav Maršálek,5,6 Jussi Meriluoto,7 Lisa Spoof7 1Department of Botany, Faculty of Biology, University of Sofia “St Kliment Ohridski”, bld Dragan Zankov 8, BG-1164, Sofia, Bulgaria; 2Unité d’Océanographie Chimique, Université de Liège, Sart Tilman, B-4000, Liège, Belgium; 3Research Unit in Organismal Biology (URBE), University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium; 4National Centre of Public Health and Analyses, Str. Academik Ivan Evstratiev Geshov 15, 1431 Sofia, Bulgaria; 5Department of Experimental Phycology and Ecotoxicology, Institute of Botany, Czech Academy of Sciences, Lidická 25/27, 602 00 Brno, Czech Republic; 6RECETOX - Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic; 7Department of Biochemistry, Faculty of Science and Engineering, Ǻbo Akademi University, Turku, Finland *Corresponding author: mstoyneva@uni-sofia.bg ABSTRACT The scientific and public awareness of hazardous photosynthetic prokaryotes (cyanobacteria/cyanoprokaryotes) and especially the contamination of drinking-water reservoirs with cyanotoxins is world-wide increasing. Recently much more attention has been paid to the events and results of mass proliferation of these toxic organisms even in South-East European countries in spite of the fact that, as a rule, they are not controlled by national legislation.The present paper presents a summary of results of such studies carried out in summer-autumn periods of the last 15 years (2000-2015) in Bulgarian water bodies differing by location, morphometry and trophic status, incl. drinking-water reservoirs, recreational lakes and sites of nature conservation importance. A multivariate analysis allowed to outline the distribution patterns and environmental drivers of the planktonic cyanoprokaryote assemblages in relation with the available data on the water bodies, highlighting species composition and abundance of the main taxa, including potentially toxic species. Samples analysis by HPLC-DAD and/or LC/MS, ELISA and in vitro cytotoxicity tests allowed detection of microcystins, nodularins and saxi- toxins. Toxin concentration ranged between 0.1 and 26.5 µg L–1 in water samples and between 10.9 and 1070 µg g–1 (d.w.) in concentrated (net) samples. Despite the fact that microcystins were not found in all studied water bodies and that the recorded levels were still lower in comparison with some other European countries, the fact that cyanotoxins were detected in 16 water bodies (incl. 3 drinking-water reservoirs) could serve as an alert for the need of recognition of cyanotoxins as a new health risk factor in the country. Therefore, per- manent monitoring with identification of toxins in water bodies at risk and activities for limitation and control of toxic blooms are ur- gently needed, in combination with increase of the attention to the effects of cyanotoxins on both human health and health of aquatic ecosystems in Bulgaria. Key words: Microcystins; nodularins; saxitoxins; reservoirs; lakes; health risk. Received: 30 September 2016. Accepted: 19 December 2016. No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.132 hazardous for human and ecosystem health (Carmichael 1994; Codd 1994, 1995; Chorus and Bartram, 1999; Codd et al., 1999, 2005a, 2005b; Maršálek et al., 2000; Huis- man et al., 2005; Meriluoto and Codd, 2005; Metcalf and Codd, 2012; Walker, 2015). The situation in Bulgaria is not an exception in this case. Moreover, the country is of intermediate position on the Balkan Peninsula - a great European hotspot of biodiversity (Griffiths et al., 2004). The studies of its water inhabitants have been conducted since more than hundred years and 216 taxa (incl. 170 cyanoprokaryotes and algae), new for the science, have been described (Michev and Stoyneva, 2007). However, the inventory assessment of Bulgarian water bodies (WBs) brought to the recognition of anthropogenically fast eutrophication with nuisance blooms in rapidly in- creasing numbers of WBs. This situation was considered by the authors as alarming. Since then the number of stud- ies and publications on the cyanotoxins in Bulgarian wa- ters, pioneered by Pavlova et al. (2006), increased and proved their existence in different sites, some of which of high conservational, drinking-water, sport fishing or recreational importance. A part of the results has been published in Bulgarian language, which, in spite of being accepted as one of the official languages of the European Union, remains exotic and not understandable for the ma- jority of scientists. Therefore, the aim of the present paper is to summarize data on cyanotoxins and water blooms in Bulgaria gathered during the last 15 years’ period and to assess them on the background of cyanoprokaryote dis- tribution, diversity and abundance in relation to potential toxin producers and driving environmental variables. METHODS Study area The study area covers practically the whole territory of Bulgaria (111,000 km2), a part of the eastern Balkan Penin- sula with The Danube and Black Sea as north and east bor- ders (Fig. 1). One-third of the country territory is occupied by plains, while the other is covered by hills, plateaus and higher mountains. Its peak Mousala (2925 m) is the highest Fig.1. Map of Bulgaria with indication of location of the 120 water bodies studied in 2000-2015. Numeration follows Tab. 1. No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 133 point of the Balkan Peninsula, while the lowest parts of Bulgaria are at sea level. The climate is temperate conti- nental with a Mediterranean influence in its southern (mainly SW) part with a significant amount of snowfall during winter. Temperature amplitudes vary in different areas (from -38.3°C to 45.2°C) and precipitation range is from 500 mm in plains to more than 2500 mm in the moun- tains, being about 630 mm per year on average. Due to the interaction of climatic, hydrological, geological and topo- graphical conditions, Bulgaria is one of the countries with highest biodiversity in Europe (Peev et al., 2013). Accord- ing to the Appendix № ХІ, map А of Water Framework Di- rective 2000/60/ЕС (European Commission, 2000) Bulgaria belongs to the ecoregions № 12 Pontic province and № 7 Eastern Balkans, the borders of which have been updated by Cheshmedjiev et al. (2010b). The results from the first Inventory of Bulgarian wetlands and their biodi- versity lead to the conclusion that the country is character- ized by great diversity and number of WBs, which cover ca. 1% of its territory (Michev and Stoyneva, 2007). The term wetlands was consistently used by these authors for all types of WBs, following the definition of Ramsar Con- vention on wetlands, signed in 1971. Data collection on planktonic cyanoprokaryotes This study compiles and evaluates the currently avail- able data on phytoplankton and relevant environmental data on Bulgarian WBs (mainly reservoirs and lakes) gathered during the period 2000-2015 (Stoyneva, 2003, 2010, 2014, 2015; Beshkova and Botev, 2004; Kalchev et al., 2004; Traykov, 2005; Pavlova et al., 2006, 2013a, 2014, 2015; Beshkova et al., 2008a, 2008b, 2012; Pavlova, 2007; Tsanev and Belkinova, 2008; Teneva et al., 2009, 2010a, 2010b, 2011, 2014; Cheshmedjiev et al., 2010a, 2013; Stoyanov et al., 2012, 2013, 2016; Stoyanov, 2014 Stoyneva et al., 2013, 2015; Belkinova et al., 2014; Dim- itrova et al., 2014a, 2014b; Dochin and Stoyneva, 2014, 2015; Dochin, 2015; Georgieva et al., 2015). The total number of WBs investigated in the above mentioned studies is 115 (Tab. 1, Fig. 1). In addition, five recreational lakes have been checked for cyanotoxins, but data on their phytoplankton composition have not been published (Pavlova, 2007; Pavlova et al., 2013a). They are added in Tab. 1 and Fig. 1 with numbers 116-120. In Tab. 1 we provide the commonly used categories (reser- voirs, lakes, swamps, etc.) and the unique number of each WB according to the DataBase of Bulgarian wetlands in- ventory (IBW - Michev and Stoyneva, 2007), where more details on the origin, location, morphometry, trophic sta- tus, history, way of exploitation, conservation, etc. could be found. Since most investigated WBs are reservoirs, in the text bellow, in an operational way, we assign all WBs to two types – reservoirs (R) and other, mainly natural WBs (OWB). According to the geographical location and vertical position all studied WBs could be grouped in the following 8 groups (phyla) of the hierarchical Bulgarian wetland classification (Michev and Stoyneva, 2007): Black Sea coastal surface lowland WBs (CV.I, 0-200 m asl) – 11 (3 R, 8 OWB); Black Sea coastal surface low mountain WBs (CV.IV; >1000 m asl) – 1 R; Inland surface lowland WBs (LV.I; 0-200 m asl) – 41 (35 R, 6 OWB); In- land surface plain WBs (LV.II; 200-600(700) m asl) – 24 R; Inland surface kettle WBs (LV.III; 500-1000 m asl– 11 (8 R, 3 OWB); Inland surface low mountain WBs (LV.IV; >1000 m asl) – 10 R; Inland surface middle mountain WBs (LV.V; 1000-1800 m asl – 3 (2 R, 1 OWB); Inland surface high mountain and alpine WBs (LV.VI; >1800 m asl – 19 (2 R, 17 OWB). Among the WBs studied 36 are of conservational importance (Tab. 1): 21 are included in the Red List of Bulgarian wetlands as critically endan- gered (CR) – 9, endangered (EN) – 6 and vulnerable (VU) – 6 (Michev and Stoyneva, 2005, 2007); 36 belong to pro- tected areas with different International and National sta- tus (Michev and Stoyneva, 2007); 17 are special subjects of National Action Plan for conservation of wetlands of high significance of Bulgaria for 2013-2022 (Vassilev et al., 2013). Phytoplankton sampling and laboratory processing Phytoplankton sampling procedures and measurements of the main environmental variables (water temperature - t, pH, electric conductivity - cond, Secchi depth - SD, total phosphorus - TP and total nitrogen -TN) were based on In- ternational and Bulgarian standards (e.g., CEN EN 15204, 2006; State Order N 4/14.09.2012, 2013; Belkinova and Gecheva, 2013 and references therein). Some studies in- cluded more environmental data (chlorophyll a, dissolved oxygen, saturation, NO3, PO4, etc.) with tools and protocols described in detail in the relevant publications, but all of them generally followed the same design. More differences concern the microscopic processing of the samples because the cited standards provide possibilities to choose between inverted microscopy and standard microscopy in combina- tion with different types of counting chambers. In the works of the authors of this paper standard light microscopy was used in combination with Thoma or Burker counting cham- bers. Always cell was the main counting unit. The biomass estimations were based on the measurement of the dimen- sions of each cell according to the method of stereometrical approximations (Rott, 1981) instead of the often recom- mended and broadly applied by other Bulgarian authors usage of average cell sizes. The reasons for choosing this more time-consuming way of work were described in Stoyneva et al. (2015) but for the purpose of the present paper they could be briefly summarised as follows: i) ne- cessity of cell measurements for correct taxa identification; ii) differences in cell size during the cell division process; 3) variations in cell size of the same taxon in different WBs, No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.134 Tab. 1. Bulgarian WBs studied in the period 2000-2015. The names are provided according to the Bulgarian Wetlands Inventory (IBW) through transliteration of their vernacular names; in brackets the synonyms used in the literature. Number Name Type GL AZ PA RL NAP IBWnumber 1 Aheloy* R CV.IV. 3 IBW3032 2 Aleksandrovo R LV.I. 3 IBW2017 3 Aleksandur Stamboliyski* R LV.I. 3 IBW2056 4 Blato Alepu* S CV.I. 1 x CR IBW1770 5 Musalensko ezero 3 (Alekovo ezero) L LV.VI. 13 x VU IBW0078 6 Antimovo* R LV.I. 3 IBW 2818 7 Asenovets R LVII 4 IBW2549 8 Asparuhov val* R LV.I. 2 IBW 3674 9 Atanasovsko ezero* L CV.I. -1 x VU x IBW1900 10 Baniska R LV.I. 3 IBW9042 11 Batak* R LV.V. 8 IBW1316 12 Bebresh* R LV.II. 5 IBW2397 13 Belmeken R LV.VI. 10 IBW1187 14 Blatse do AEC-Belene TS LV.I. 1 x IBW4638 15 Beli Iskur* R LV.VI. 10 x IBW1180 16 Beli Lom R LV.II. 5 IBW2810 17 Bezbozhsko ezero 1 (Ezero Bezbog)* L LV.VI. 11 x IBW0442 18 Balastrierni ezera Bistratsite/Bistraka L LV.III. 5 IBW4563 19 Bistritsa* R LV.III. 6 IBW1067 20 Blatse do AEC-Kozloduy T LV.I. 1 21 Borovitsa R LV.IV. 7 IBW1580 22 Boyka R LV.II 4 IBW2573 23 Vaya (Burgasko ezero)* L CV.I. 1 x CR x IBW0191 24 Barzina* R LV.I. 3 IBW1276 25 Nevenino ezero 1 (Chernoto ezero)* L LV.VI. 12 x IBW0371 26 Chirpan* R LV.I. 3 IBW1704 27 Choklyovo Blato* S LV.V. 7 x VU x IBW 0003 28 Gergiysko ezero 2* L LV.VI. 12 x IBW0480 29 Gorni Dabnik* R LV.I. 3 IBW 5606 30 Daskal Atanasovo R LV.I. 3 IBW2219 31 Devets (Monchovets) R LV.II 4 IBW10869 32 Dospat* R LV.V. 5 IBW3155 33 Drenovets R LV.I. 3 IBW1128 34 Dabnika R LV.II. 5 IBW5393 35 Durankulashko ezero (Durankulak)* L CV.I. 1 x CR x IBW0216 36 Dyakovo* R LV.IV. 6 IBW1033 37 Blato Dyuleva Bara T LV.I. 1 x EN x IBW0154 38 Eleshnitsa* R LV.I. 2 IBW3023 39 Enitsa R LV.I. 3 IBW1444 40 Ivaylovgrad* R LV.I. 3 IBW2271 41 Iskur* R LV.III. 7 IBW1200 42 Hr. Smirnenski na reka Lom* R LV.I. 3 IBW1135 43 Hr. Smirnenski na reka Yantra (Hr. Smirneski/Gabrovo)* R LV.II. 5 IBW2080 44 Kamenets R LV.I. 3 IBW2162 45 Karaisen R LV.I. 3 IBW5113 46 Musalensko ezero 7 (Karakashevo ezero) L LV.VI. 13 x VU IBW0080 47 Seyatchi (Kavacite, Popovo)* R LV.II. 4 IBW2606 48 Kamchiya* R LV.IV. 5 IBW2745 49 Koprinka* R LV.II. 5 IBW2062 50 Kovachitsa R LV.I. 3 IBW1160 51 Krapets* R LV.III. 5 IBW2000 52 Kremensko ezero 2* L LV.VI. 12 x IBW9088 To be continued on next page No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 135 Tab. 1. Continued from previous page. Number Name Type GL AZ PA RL NAP IBWnumber 53 Krichim R LV.I. 3 IBW1366 54 Krushovitsa 3 (Krushovitsa) R LV.I. 1 IBW1452 55 Kula R LV.II. 4 IBW1105 56 Kardzhali* R LV.II. 5 IBW1668 57 Musalensko ezero 1 (Ledeno ezero) L LV.VI. 13 x VU IBW0076 58 Lazhenska bara (Ladzhenska bara) R LV.I/ 2 IBW2166 59 Mandra* R CV.I. 1 x EN x IBW1720 60 Marichino ezero 2 (Gorno Marichino ezero) L LV.VI. 12 x IBW0085 61 Marichino ezero 3 (Dolno Marichino ezero) L LV.VI. 12 x IBW0086 62 Murtvo blato TS LV.I. 1 x EN x IBW0158 63 Ezero Momin brod* L LV.I. 1 IBW8307 64 Novo Zhelezare R LV.II. 4 IBW1475 65 Ognyanovo* R LV.III. 6 IBW2340 66 Ogosta* R LV.I. 3 IBW 1137 67 Ovcharitsa R LV.I. 3 x x IBW2317 68 Ovchi kladenets R LV.I. 3 IBW2367 69 Pchelina* R LV.III. 6 IBW1039 70 Blato Peschina (Pischina) T LV.I. 1 x EN x IBW0156 71 Poletkovtsi 2 (Poletkovtsi)* R LV.II. 4 IBW1103 72 Pomoriysko ezero* L CV.I. -1 x VU x IBW 0189 73 Popovo ezero 2 (Popovo ezero)* L LV.VI. 11 x IBW0447 74 Poroy* R CV.I. 2 IBW3038 75 Pyasuchnik 1 (Pyasuchnik)* R LV.II. 4 x IBW5834 76 Rabisha* R LV.II. 4 IBW1102 77 Rasovo 2 (Rasovo) R LV.I. 3 IBW1158 78 Redzhepsko ezero 2 (Redzhepsko ezero) R LV.VI. 12 x IBW0342 79 Dragash Voyvoda R LV.I. 1 x IBW3935 80 Onogour (Efreytor Bakalovo) R LV.I. 3 x IBW5667 81 Bunderishko ezero 9 (Ribno ezero)* L LV.VI. 11 x IBW0400 82 Shablensko ezero (Shabla)* L CV.I. 1 x CR x IBW0219 83 Shablenska tuzla L CV.I. -1 EN IBW0218 84 Kayabash 2 (Golyamo Skalensko ezero) R LV.IV. 5 IBW2659 85 Kayabash 1 (Malko Skalensko ezero) R LV.IV. 5 IBW2658 86 Hisar 12 (Sinyata reka)* R LV.II. 5 IBW1893 87 Sopot* R LV.II. 5 IBW1437 88 Blato Srebarna* L LV.I. 1 x EN x IBW0208 89 Srechenska bara* R LV.IV. 5 IBW3668 90 Stoychovtsi (Stoykovtsi) R LV.IV. 8 IBW3237 91 Studena* R LV.IV. 7 IBW 1060 92 Studen kladenets* R LV.II. 4 IBW1763 93 Suedinenie* R LV.I. 3 IBW2642 94 Telish* R LV.I. 3 IBW1413 95 Ticha* R LV.I. 3 IBW2700 96 Toshkov chark R LV.V. 9 x IBW1315 97 Trakiets R LV.II. 4 IBW1677 98 Tri kladentsi* R LV.I. 3 IBW1275 99 Tsonevo* R LV.I. 2 IBW3022 100 Varnensko ezero* L CV.I. 1 x CR IBW0203 101 Vlahinsko ezero 1* L LV.VI. 12 x IBW0475 102 Vucha R LV.I. 3 IBW 3143 103 Valchovets R LV.I. 2 IBW2129 104 Yarlovets (Yarlovtsi) R LV.IV. 7 IBW 1038 105 Yasna polyana* R CV.I. 2 IBW2887 To be continued on next page No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.136 or even in the same water body due to different tempera- ture, nutrient content or grazing pressure (Stoyneva et al., 2007). We believe that the results obtained in this way re- flect the real biomass for a given time and site, especially when the samples are analysed by the same person. Multivariate analyses Considering the reasons stated above, after analyzing of all available, but quite heterogenous data on the 2000- 2015 phytoplankton in Bulgarian WBs (references pro- vided in the “Data Collection” paragraph above), we chose to build a homogenous data set (Stoyneva, 2014, 2015; this study) by including only the WBs (sites) with at last 5 samples processed per site, for which the envi- ronmental data listed above were available. WBs in which cyanotoxins have been investigated by the authors of this paper, have been included in the dataset. In addition, WBs were chosen to represent all 8 general groups (from CV.I to LV.VI) proposed in the Bulgarian wetlands classifica- tion and the CR, EN and VU conservational threat cate- gories (Michev and Stoyneva 2005, 2007) as well. In this way, the final dataset comprised 61 WBs (marked with asterisk* in Tab. 1) and average values of biomass of 93 species, varieties and forms grouped in 24 genera, envi- ronmental data, total cyanoprokaryote biomass (TBC), total phytoplankton biomass (TBS). The dataset also in- cludes average data biomass of other phytoplankton dom- inant groups, as well as data on WBs morphometry (area and depth), geographic location in the 8 groups of Michev and Stoyneva (2007), exact altitude asl (m) or as 14 ele- vation classes proposed in the IBW DataBase of Michev and Stoyneva (2007): -1 – below 0; 1-0-50; 2 – 50-100; 3 – 100-200; 4 – 200-300; 5 – 300-500; 6 – 500-700; 7 – 700-1000; 8 – 1000-1100; 9 – 1100-1600; 10 – 1600- 2000; 11 – 2000-2300; 12 – 2300-2700; 13 – >2700 m asl. Multivariate analyses were run using principal com- ponents analyis (PCA) and redundancy analysis (RDA), using the R-software (R 3.1.2 version, R Development Core Team, 2010) and the “ADE4” package (Thioulouse et al., 1997). The aims were i) to identify the main envi- ronmental gradients among the samples of lakes/reser- voirs and ii) to investigate the response of the cyanoprokaryote assemblages (at the genus level) to these gradients. All variables were normalised; the cyanobacte- rial abundances were transformed using the Hellinger transformation (Legendre and Gallagher, 2001). Taxonomic sources, terminology and biodiversity assessment Taxonomic sources include mainly the standardly used volumes of Middle European freshwater flora with some published updates (Komárek 2013; Komárek and Anagnos- tidis 1999, 2005; Komárek et al. 2011, etc.) and AlgaeBase (Guiry and Guiry 2016). The Latin names follow the above mentioned botanical sources and the International Code of the Nomenclature for algae, fungi and plants (McNeil et al., 2012). Therefore, the term cyanoprokaryotes is used in Tab. 1. Continued from previous page. Number Name Type GL AZ PA RL NAP IBWnumber 106 Yastrebino* R LV.II. 5 IBW2602 107 Shilkovtsi (Yovkovtsi)* R LV.II. 5 IBW2105 108 Zhrebchevo* R LV.II. 4 IBW2545 109 Lomtsi R LV.II. 4 IBW2772 110 Pancharevo R LV.III. 6 IBW1088 111 Zhernov R LV.I. 2 IBW4639 112 Ezero Bliznaka L LV.VI. 11 x CR x IBW0350 113 Ezero Bubreka L LV.VI. 11 x CR x IBW0349 114 Ezero Okoto L LV.VI. 12 x CR x IBW0348 115 Ezero Sulzata L LV.VI. 12 x CR x IBW0347 116 Balastrierni ezera Dolni Bogrov L LV.III. 6 IBW0708 117 Botunets R LV.III. 6 IBW1792 118 Ezero 1 v kvartal Druzhba L LV.III. 6 IBW5284 119 Krasava R LV.IV. 7 IBW1049 120 Rudnichno ezero Кutina 1 L LV.III. 6 IBW0705 *Denote WB included in the dataset for the Principal component analysis (PCA) and Redundancy analysis (RDA) for this paper. R, reservoir; L, lake; S, swamp; T, temporary swamp. For each WB, the main group of geographic location and vertical position (GL) is shown (CV.I – LV.VI) and altitude zone (AZ) in 14 elevation classes (-1 -13); for details on GL and AZ see the text. The conservation value of the WB is provided as protected area (PA), National Action Plan for conservation of wetlands of high significance of Bulgaria for 2013-2022 (NAP) and Red List of Bulgarian Wetlands (RL); CR, critically endangered; EN, endangered; VU, vulnerable. IBW number is the number of WB in the Bulgarian Wetlands Inventory (Michev and Stoyneva, 2007), where more details on them could be found. No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 137 the paper instead of cyanobacteria. For each species and genus, the frequency quotient (FQ) of occurrence in all studied sites was estimated based on its presence / absence in each site. The frequency quotients were grouped in classes with a step of 10% (I class – 0-10%, II class – 10- 20%, etc.). The evaluation of cyanoprokaryote diversity was done as comparisons with the recently estimated data on the total algal biodiversity in Bulgaria (Stoyneva, 2014) and the total diversity of Cyanoprokaryota in the country (Stoyneva et al., 2016). Collection and assessment of cyanotoxin data, algal blooms and potential toxin producers The assessment of cyanotoxins registered in the coun- try in relation to their potential producers is based on the works of Pavlova (2007), Pavlova et al. (2006, 2007, 2013a, 2014, 2015), Teneva et al. (2009, 2010a, 2010b, 2011, 2014), Stoyanov et al. (2012) and Georgieva et al. (2015) – Tabs. 2 and 3. For this study the biomass of the species published earlier by Pavlova et al. (2006, 2014) with their cell numbers per liter (or milliliter) was esti- mated (Tabs. 2 and 3). Results on toxin findings were su- perimposed on the data on phytoplankton blooms according to the references in Tabs. 2 and 3. The cyan- otoxin concentrations were evaluated according to the WHO standards (1998, 2003) since their maximum ac- ceptable levels are not indicated in Bulgarian national leg- islation (Pavlova et al., 2013b). RESULTS Diversity and abundance of cyanoprokaryotes A total of 210 taxa (207 species, 1 variety and 2 forms) from 69 genera of Cyanoprokaryota were recorded in the 2000-2015 period. The distribution of species in the three orders Chroococcales, Oscillatoriales and Nostocales clearly shows the Chroococcales (85) as the richest order and the better representation of non-heterocytous filamen- tous forms (69) in comparison with hetereocytous, fila- mentous taxa (56). At the genus level, heterocytous taxa (17) were also less numerous than non-heterocytous fila- mentous (21) and than coccal taxa (31). The most species- rich genera were Dolichospermum (11), Microcystis (11), Anabaena (9), Aphanocapsa (9), Oscillatoria (9), Chroococcus (8), Phormidium (8), Pseudanabaena (8), Romeria (8), Anabaenopsis (6) and Aphanizomenon (6). Most of the species were rare (96 were found only in one site) and FQ ranged from 1 to 34%. Among the 19 broadly distributed algae (in ≥11 sites), 14 have been iden- tified at species level (Fig. 2), with Aphanizomenon flos- aquae Ralfs ex Bornet & Flahault being the most widespread taxon. Most of the 69 genera were rare (18 were found in one site), with FQ range from 1 to 43%. The 22 genera with the widest distribution (in ≥12 sites) are shown on Fig. 3: Aph- anizomenon is outstanding as the most widespread genus in Fig. 2. Distribution of the most widespread cyanoprokaryotes in the phytoplankton of Bulgarian water bodies in the period 2000-2015. 0-40, number of water bodies in which species were found; *species found in the samples containing cyanotoxins. No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.138 Ta b. 2 .C ya no to xi ns in B ul ga ri an W B s, o rg an iz ed a cc or di ng to th e fi rs t y ea r o f f in di ng . W at er b od y M ai n us e D at e B io m as s M C (t yp es ) M C to ta l N O D A N T- a S T X M et ho ds S ou rc e L R R R Y R L A Y R eq M C /N O D N O D H P L C E lis a I V C T B is tr its a R S 2 0. 8. 20 04 1 .5 3* 0 .8 6 x x 1. 64 x Pa vl ov a et a l. 20 06 14 .1 0. 20 13 n. a. 0 .6 0 .6 x Pa vl ov a et a l. 20 15 B or ov its a D R 2 6. 7. 20 06 n ot in d. 0 .0 9- 0. 12 x x x T en ev a et a l. 20 10 b 2 6. 9. 20 06 n ot in d. x x x x 0 .1 8 2 .5 x x T en ev a et a l. 20 10 b V uc ha W S A ug 20 08 0 .1 1 0 .0 04 x x x Te ne va e t a l. 20 10 a S ep t2 00 9 0 .0 62 0 .0 05 x x x Te ne va e t a l. 20 10 a St ud en k la de ne ts -d am w al l p ar t IR Ju ly 20 08 n ot in d. 0 .2 -0 .4 x x Te ne va e t a l. 20 11 A ug 20 08 0 .0 7 0 .2 -0 .4 x x T en ev a et a l. 20 11 S ep t2 00 8 0 .1 0 .2 -0 .4 x x T en ev a et a l. 20 11 J ul y2 00 9 0 .0 21 0 .2 -0 .4 x T en ev a et a l. 20 12 A ug 20 09 0. 01 1 0 .2 -0 .4 x x x T en ev a et a l. 20 11 S ep t2 00 9 0 .0 33 0 .2 -0 .4 x <0 .1 0 x x Te ne va e t a l. 20 11 St ud en k la de ne ts - ta il pa rt J ul y2 00 8 n ot in d. 0 .2 -0 .4 x x Te ne va e t a l. 20 11 A ug 20 08 0 .1 9 0 .2 -0 .4 x x T en ev a et a l. 20 11 S ep t2 00 8 0 .1 0 .2 -0 .4 x x T en ev a et a l. 20 11 J ul y2 00 9 0 .0 94 x < 0. 10 x x T en ev a et a l. 20 11 A ug 20 09 0. 76 1 x < 0. 10 x x T en ev a et a l. 20 11 S ep t2 00 9 0 .4 53 0 .2 -0 .4 x x x Te ne va e t a l. 20 11 Tr ak ie ts D R Ju ly 20 08 0 .5 x x x x 0. 09 x x x x Te ne va e t a l. 20 09 A ug 20 08 0 .0 4 x x x x 0 .0 18 x 0 .0 1 x x x Te ne va e t a l. 20 09 S ep t2 00 9 0. 06 x x x x 0 .0 14 x x x x Te ne va e t a l. 20 09 K ru sh ov its a R S 4 .1 0. 20 09 2. 32 1 x x x S to ya no v et a l. 20 12 E ni ts a R S 4 .1 0. 20 09 1 4. 57 2 x x x S to ya no v et a l. 20 12 V al ch ov et s W S 4 .1 0. 20 09 0 0 .0 2 x x x S to ya no v et a l. 20 12 Pc he lin a R S 3. 8. 20 11 0. 16 7 0 .5 0 .5 x Pa vl ov a et a l. 20 14 2 .1 0. 20 14 6 .2 0 .1 7 0 .0 7 0. 13 0. 37 x Pa vl ov a et a l. 20 15 K ay ab as h 2 IR Ju ly 20 11 3 0 0 .4 x x x Te ne va e t a l. 20 14 S ep t2 01 1 6 5. 42 0. 4 x x x Te ne va e t a l. 20 14 St ud en a D R 2 8. 9. 20 11 0. 1 0 .1 x Pa vl ov a et a l. 20 14 14 .1 0. 20 13 0. 1 0 .1 P av lo va e t a l. 20 15 5 .1 1. 20 15 0 .0 04 * 0 .2 0 .4 0 .6 x G eo rg ie va e t a l. 20 15 E ze ro M om in b ro d R S 1 9. 8. 20 12 0 .8 1 0 .3 1 .3 x P av lo va e t a l. 20 15 21 .1 0. 20 13 3 .7 1 1 0 .4 0 .4 1 .8 x Pa vl ov a et a l. 20 15 D ur an ku la sh ko e ze ro R S 1 8. 8. 20 13 10 .5 0 .2 0 .1 0 .9 1 .2 x Pa vl ov a et a l. 20 15 2 9. 7. 20 11 7. 01 8 .3 12 .7 5 .5 2 6. 5 x P av lo va e t a l. 20 14 1 8. 8. 20 13 10 .5 0 .2 0 .1 0 .9 1 .2 x Pa vl ov a et a l. 20 15 N et s am pl es / sc um “ bi om as se s” V ay a R S 5. 8. 20 04 6 8. 56 * 1 4 x x 4 2 x Pa vl ov a et a l. 20 06 V ay a -q ua y 5 .8 .2 00 4 3 2. 34 * 2 60 x x 1 07 0 x Pa vl ov a et a l. 20 06 D ur an ku la sh ko e ze ro R S 3. 8. 20 04 5. 43 * 2 60 11 0 x Pa vl ov a 20 07 ; P av lo va et a l. 20 06 . 2 00 7; 1 4. 7. 20 05 n .a . 2 04 x x 51 7 x P av lo va e t a l. 20 07 , 2 01 3 2 9. 7. 20 11 n .a . 2 7. 5 2 2. 1 x( tr ac e) 49 .6 P av lo va e t a l. 20 14 1 8. 8. 20 13 n .a . 6 3. 5 10 3. 2 4 7. 6 2 14 .3 x Pa vl ov a et a l. 20 15 To b e co nt in ue d on n ex t p ag e No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 139 the country during the analyzed period. In this study, we classified as picoplankton single spherical cells of dimen- sions 0.5-1.5 µm, commonly referred as Pcy (picocyanobac- teria) in contrast to the second morphological group of colonial picoplankters – CPcy (colonial picocyanobacteria) – Stockner et al. (2002), Callieri et al. (2012). The number of species and genera per site ranged be- tween (0)1 and 85, and 1 to 35, respectively (Fig. 4), the highest number detected in the shallow lakes Blato Sre- barna (site 88: 85/35) and Vaya (site 23: 63/30). The com- parison of distribution of species and genera per site, expressed on Fig. 4, shows that in most of the WBs each genus is represented by a single species, which makes the discussion of the distribution on generic level and further PCA analysis quite reasonable. The contribution of the cyanoprokaryotes to the total phytoplankton biomass in different WBs and in different sampling periods ranged from 0 to 100%, with values ex- ceeding 65-75 mg L–1 in summer periods (e.g., in the coastal lake Vaya - Stoyneva, 2003; Pavlova et al., 2006, 2007; Dimitrova et al., 2014a) and reaching 95.9 mg L–1 in average for the period 2010-2014 (this study). The as- sessment of the average cyanoprokaryote biomass in 61 WBs from the dataset shows that in four sites (the lakes Vaya and Blato Srebarna, and the reservoirs Mandra and Tri kladentsi), its values were over WHO’s (2003) thresh- old for medium health risk category (10 mg L–1) in recre- ational waters and in five other (the lakes Durankulashko ezero and Ezero Momin brod, and the reservoirs As- paruhov val, Chirpan and Pchelina) from the rest 57 WBs, they were over the low health risk category threshold (2 mg L–1), as they were re-estimated from cell numbers in biomass values by Mishke et al. (2011). The single investigation of steady-states, as Sommer and Padisák (1993) have defined them, outlined their pres- ence in the period analyzed for this paper only in the lake Vaya: 3 weeks dominance (89%) of Microcystis wesen- bergii (Komárek) Komárek in Kondratieva, Aphani- zomenon flos-aquae and Dolichospermum spiroides (Klebahn) Wacklin et al. in August–September 2001 and 4 weeks dominance (98%) of M. wesenbergii and Aphani- zomenon gracile (Lemmermann) Lemmermann in August- September 2002 (Stoyneva, 2003). The lake Vaya is the only WB for which the total carbon content was estimated (Dimitrova et al., 2014a). Its mean value of 9.7 mg L–1 (2004-2006) together with the average biomass of 46 mg L–1 for the same period confirmed the hypertrophic status of the lake. Cyanoprokaryotes dominated constantly in the total carbon content, reaching absolute maxima of 25.1 and 26.9 mg L–1 in August 2005 and 2006. Dominance of this group with water blooms (up to 200 mg L–1 ) was detected also in more recent studies of this shallowest coastal lake and, in parallel, cyanoprokaryote blooms were documented for 19 more Bulgarian WBs: Alepu, Durankulashko ezero,Ta b. 2 .C on tin ue d fr om p re vi ou s pa ge . W at er b od y M ai n us e D at e B io m as s M C (t yp es ) M C to ta l N O D A N T- a S T X M et ho ds S ou rc e L R R R Y R L A Y R eq M C /N O D N O D H P L C E lis a I V C T N et s am pl es / sc um “ bi om as se s” M an dr a IR . R S 6. 8. 20 04 2. 61 * 35 x x 6 3 x Pa vl ov a et a l. 20 06 Pc he lin a IR 19 .8 .2 00 4 0 .0 8* 1 40 x x 53 6 x Pa vl ov a et a l. 20 06 3 .8 .2 01 1 n .a . 1 1. 14 11 .1 4 x P av lo va e t a l. 20 14 3 1. 7. 20 12 n .a . x (t ra ce s) x P av lo va e t a l. 20 15 1 8. 9. 20 12 n .a . x (t ra ce s) x P av lo va e t a l. 20 15 2 .1 0. 20 14 n .a . 1 32 .8 9 1. 5 71 .8 2 96 .1 x P av lo va e t a l. 20 15 Sh ab le ns ko e ze ro R S 3. 8. 20 04 0. 62 * 40 4 0 x P av lo va e t a l. 20 06 . 2 00 7 1 4. 7. 20 05 n .a . 2 83 x x x 1 01 8 x P av lo va 2 00 7; P av lo va e t a l. 20 07 Sh ab le ns ko e ze ro - qu ay 3 .8 .2 00 4 1 1. 97 * 1 4 1 4 x P av lo va e t a l. 20 06 . 2 00 7 1 4. 7. 20 05 n .a . 2 92 x x x 98 2 x P av lo va 2 00 7; P av lo va e t a l. 20 07 E ze ro M om in b ro d R S 1 9. 8. 20 12 n .a . 23 4 2 7 x Pa vl ov a et a l. 20 15 St ud en a D R 1 4. 10 .2 01 3 n .a . 8. 1 2 0 .8 10 .9 x Pa vl ov a et a l. 20 15 *B io m as s es tim at ed fo r t hi s st ud y; x , p re se nc e / n ot v al ue in di ca te d; IV C T, in v itr o cy to to xi ci ty ; n .a ., no t a na ly se d; n ot . i nd ., no in fo rm at io n in th e pu bl ic at io n; b io m as s [m g L –1 ], re la te d to th e cy an op ro ka ry ot es in th e re le va nt s am pl es ; D R , d ri nk in g- w at er r es er vo ir ; W R , r es er vo ir f or w at er s up pl ; I R , r es er vo ir f or ir ri ga tio n an d/ or e ne rg y su pp ly ; R S, r ec re at io na l s ite . C on ce nt ra tio ns a re e xp re ss ed a s µg L –1 (w at er bo dy ) o r i n µg g –1 dr y w ei gh t ( bi om as s) . No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.140 Tab. 3. Water samples with negative results for cyanotoxins in Bulgarian WBs (2000-2015). Water body -water samples checked for toxins with Date Methods Source negative results/organized by years of the investigations HPLC Elisa In vitro CT Botunets 20.8.2004 x Pavlova et al. 2006 Ezeretsko ezero 3.8.2004 x Pavlova et al. 2006 2005 x Pavlova 2007; Pavlova et al. 2013a Choklyovo blato 19.8.2004 x Pavlova et al. 2006 2005 x Pavlova 2007; Pavlova et al. 2013a Balastrierni ezera Dolni Bogrov 20.8.2004 x Pavlova et al. 2006 Ezero 1 v kvartal Druzhba 27.8.2004 x Pavlova et al. 2006 Iskur 18.8.2004 x Pavlova et al. 2006 2005 x Pavlova 2007; Pavlova et al. 2013a Rudnichno ezero Kutina 1 20.8.2004 x Pavlova et al. 2006 Studena 19.8.2004 x Pavlova et al. 2006 2005 x Pavlova 2007; Pavlova et al. 2013a 31.7.2012 x Pavlova et al. 2015 3.8.2011 x Pavlova et al. 2014 18.9.2012 x Pavlova et al. 2015 12.8.2013 x this study 2.10.2014 x Pavlova et al. 2015 4.8.2015 x Georgieva et al. 2015 Yasna polyana 6.8.2004 x Pavlova et al. 2006 2005 x Pavlova 2007; Pavlova et al. 2013a Bistritsa 2005 x Pavlova 2007; Pavlova et al. 2013a 3.8.2011 x Pavlova et al. 2014 28.9.2011 x Pavlova et al. 2014 31.7.2012 x Pavlova et al. 2015 18.9.2012 x Pavlova et al. 2015 12.8.2013 x Pavlova et al. 2015 2.10.2014 x Pavlova et al. 2015 4.8.2015 Georgieva et al. 2015 5.11.2015 x Georgieva et al. 2015 Borovitsa 2005 x Pavlova 2007; Pavlova et al. 2013a Vaya 2005 x Pavlova 2007; Pavlova et al. 2013a 3.8.2011 x Pavlova et al. 2014 2.8.2012 x Pavlova et al. 2015 16.8.2012 x Pavlova et al. 2015 21.8.2013 x Pavlova et al. 2015 Durankulashko ezero 2005 x Pavlova 2007; Pavlova et al. 2013a 12.7.2012 x Pavlova et al. 2015 8.8.2012 x Pavlova et al. 2015 Krasava 2005 x Pavlova 2007; Pavlova et al. 2013a Hristo Smirnenski na reka Yantra 2005 x Pavlova 2007; Pavlova et al. 2013a Yovkovtsi (VT) 2005 x Pavlova 2007; Pavlova et al. 2013a Mandra 2005 x Pavlova 2007; Pavlova et al. 2013a Pchelina 2005 x Pavlova 2007; Pavlova et al. 2013a 28.9.2011 x Pavlova et al. 2014 31.7.2012 x Pavlova et al. 2015 18.9.2012 x Pavlova et al. 2015 12.8.2013 x Pavlova et al. 2015 14.10.2013 x Pavlova et al. 2015 4.8.2015 Georgieva et al. 2015 5.11.2015 x Georgieva et al. 2015 Ticha 2005 x Pavlova 2007; Pavlova et al. 2013a Trakiets 2005 x Pavlova 2007; Pavlova et al. 2013a Shablensko Ezero 2005 x Pavlova 2007; Pavlova et al. 2013a Kayabsh 1 July2011 x x x Teneva et al. 2014 Sept2011 x x x Teneva et al. 2014 July2012 x x x Teneva et al. 2014 Sept2012 x x x Teneva et al. 2014 Kayabash 2 July2012 x x x Teneva et al. 2014 Sept2012 x x x Teneva et al. 2014 Ezero Momin brod 24.5.2013 x this study No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 141 Blato Srebarna and for the reservoirs Acheloy, Boyka, Borovitsa, Barzina, Daskal Atanasovo, Enitsa, Kamenets, Krushovitsa, Kardzhali, Mandra, Ovchi kladenets, Pche- lina, Seyachi, Suedinenie, Tri kladentsi, Vucha (Traykov, 2005; Cheshmedjiev et al., 2010a; Stoyanov et al., 2012; Belkinova et al., 2014; Stoyneva, 2014) (Fig. 1). The same authors and also Dochin and Stoyneva (2014, 2015) indi- cated the “presence of toxic species (Anabaena, Aphani- zomenon, Microcystis, etc.)” for the following 43 WBs: Choklyovo blato, Durankulashko ezero, Shablensko ezero, Blato Srebarna and for the reservoirs Aheloy, Aleksan- drovo, Antimovo, Asparuhov val, Batak, Boyka, Barzina, Daskal Atanasovo, Dospat, Drenovets, Dabnika, Dyakovo, Hristo Smirnenski (na reka Lom), Ivaylovgrad, Kamenets, Krapets, Krushovitsa, Koprinka, Kovachitsa, Kula, Lomtsi, Mandra, Ogosta, Ovcharitsa, Pancharevo, Pchelina, Po- letkovtsi, Poroy, Pyasuchnik, Rabisha, Rasovo, Seyachi, Suedinenie, Telish, Tri kladentsi, Valchovets, Vucha, Yas- trebino and Zhrebchevo (Fig. 1). Toxic species were indi- cated as blooming in the reservoirs Kamenets, Beli Lom and Ovchi kladenets but have not been enlisted (Cheshmed- jiev et al., 2010a) (Fig. 1). Environmental gradients and cyanoprokaryotes distribution The results of the PCA, run on the environmental vari- ables, total phytoplankton biomass (TBS) and total cyanoprokayotes biomass (TBC) of 61 WBs are shown in Fig. 5. The cumulated relative inertia of the two first prin- cipal components reached 51.4 %. The main environmental gradient, associated with the first principal component, is determined by altitude and trophic status, with altitude (alt) positively correlated with Secchi depth (SD), and nega- tively with TN and TP. Unsurprisingly, TBS and TBC is correlated with the nutrient loading. The second principal component is mainly determined by temperature and depth (Fig. 5). Three groups of WBs are identified in this analysis: group 2 is constituted essentially by 3 closely situated coastal shallow WBs (namely Atanasovsko ezero, Vaya and Mandra), which form the well-known geographical group Bourgaski ezera) and group 3, formed by clear, oligotrophic alpine lakes and one high mountain reservoir (reservoir Beli Iskur, Bezbozhsko ezero 1, Nevenino ezero 1, Gergiysko ezero 1, Vlahinsko ezero 1, Bunderishko ezero 9 and Fig. 3. Distribution of the most widespread genera of cyanoprokaryotes in the phytoplankton of Bulgarian water bodies in the period 2000-2015. 0-40, number of water bodies in which species were found; *genus found in the samples containing cyanotoxins. No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.142 Popovo ezero 2). The central group 1 is more heterogenous, comprising mostly mid-altitude WBs with varying depth and nutrient loading, but also some shallow eutrophic lakes (close to group 2, left side of the ordination). The distribu- tion of the WBs in Fig. 5 clearly shows the influence of ge- ographic location on the WBs’ characteristics. The next redundancy analysis (Fig. 6), in which the de- pendent variables are the biomass data on 24 cyanoprokary- ote genera, showed that cyanoprokaryote assemblages responded significantly to the environmental conditions, with TP as the most influential variable, followed by SD, t and TN. Four main groups of genera were identified: two (1 and 2) on the left side of the diagram, associated with eutrophic conditions in low altitude shallow lakes, swamps and reservoirs, one (group 3) of high altitude, clear lakes; and one (4) more heterogeneous group with predominance of high conductivity conditions. Within this last are asso- ciated all rare colonial non-toxic species /Rccl/ from genera like Lemmermanniela, Coelomoron, etc. and Merismopedia (mainly M. tenuissima Lemmermann) - Mrsp, non-colonial picoplankters of Pcy group – Pcpl) and Spirulina and Glau- cospira species (Spgl) with a single Phormidium (Phrm). In group 1 were Microcystis - Mcrs; Anabaena s.l. (mainly Dolichospermum) – Andl, Aphanizomenon s.l. – Aphn, Cylindrospermopsis (in this case C. raciborskii (Wołoszyńska) Seenaya & Subba Raju only) - Clps, Plank- tothrix – Plnt, Planktolyngbya s.l. (incl. Limnolyngbya) – Plnb, Romeria - Rmrr, Oscillatoria and Borzia - Osbr. Group 2 included Anabaenopsis -Anbs, Pseudanabaena – Psnb, Plectonema – Plct, Leptolyngbya – Lptb. Group 3 contains Aphanocapsa - Apns, Chroococcus - Crcr, Coelosphaerium - Clsp, Snowella - Snwl, Woronichinia - Wrnc) and Synechocystis (Sncs). Cyanotoxins The analysis of published data on cyanotoxins revealed their presence in 16 WBs – reservoirs Bistritsa, Borovitsa, Enitsa, Kayabash 2, Krushovitsa, Mandra, Pchelina, Stu- dena, Studen kladenets, Trakiets, Vucha, Valchovets and lakes Vaya, Durankulashko ezero, Ezero Momin brod and Shablensko ezero (Tab. 2, Fig. 1). During the summer-au- tumn period, microcystins LR, LA, RR, YR and similar to YR-type, nodularins, anatoxin-a and saxitoxins (from the decarbamoyl saxitoxin, gonyautotoxins II, III, B1, C1 and C2 group) in different concentrations were proved by High Performance Liguid Chromatography (HPLC, HPLC-DAD and/or HPLC-MS), enzyme-linked immunosorbent assay (ELISA) and in vitro cytotests (Tab. 2). Anatoxin was de- tected only once, in July 2006 in Borovitsa reservoir by HPLC (Teneva et al., 2009). Saxitoxins (STXs) were found in the reservoirs Borovitsa, Studen kladenets, Trakiets and Fig. 4. Distribution of the number of species and genera of cyanoprokaryote phytoplankters in 115 Bulgarian water bodies in the period 2000-2015. No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 143 Fig. 5. Results of the principal component analysis on the environmental variables and phytoplankton/cyanoprokaryote biomass of 61 Bulgarian water bodies (WBs); ordination on the two first components (cumulative inertia: 51.4%). Numbers and names of the WBs as in Tab. 1. t, water temperature; cond, electric conductivity; SD, Secchi depth; TP, total phosphorus; TN, total nitrogen. Fig. 6. Results of the redundancy analysis on the environmental variables and cyanoprokaryote genera of 61 Bulgarian water bodies (WBs); ordination on the two first components (cumulative inertia: 45.3%). Numbers and names of the WBs as in Tab. 1. ha, area; alt, altitude; t, water temperature; cond, electric conductivity; SD, Secchi depth; TP, total phosphorus; TN, total nitrogen. No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.144 Valchovets by Teneva et al. (2009, 2010a, 2011) and Stoy- anov et al. (2012). Their concentrations varied between 0.1 and 2.5 μg L–1, except in Studen kladenets, where STXs were detected only by HPLC peaks and authors sug- gest that their concentrations in the water were less than their mean lower detection limit by Ridascreen™ assay – 0.01 μg L–1. In the same reservoir HPLC peaks showed the presence of nodularins, but their amounts were given only as a total of microcystins/nodularins (MC/NOD) detected by ELISA (Teneva et al., 2011). In the same way, MC/NOD totals were provided for the reservoirs Borovitsa, Enitsa, Kayabash 2, Krushovitsa, Studen kladenets and Trakiets (Teneva et al., 2009, 2010a, 2011, 2014; Stoyanov et al., 2012). The types of microcystins were indicated in the publications for 10 WBs but only for 9 of them the values have been provided (Tab. 2). Accord- ing to Pavlova et al. (2006, 2007, 2013a, 2014), Pavlova (2007) and Georgieva et al. (2015) the concentrations of the different microcystin types ranged as follows: LR – 0.1-8.3 μg L–1 in water samples and 14-292 μg g–1 in net samples, RR - 0.1-12.7 μg L–1 in water samples and 2- 103.2 μg g–1 in net samples, YR – 0.13-5.5 μg L–1 in water samples and 0.8-71.8 μg g–1 in net samples. YR equivalent type was detected twice by HPLC-DAD in Shablensko ezero (Pavlova, 2007; Pavlova et al., 2007) and LA type was reported once from Borovitsa by Teneva et al. (2010b). Negative results for cyanotoxins have been published by Pavlova (2007), Pavlova et al. (2007, 2013a, 2014, 2015), Teneva et al. (2014) and Georgieva et al. (2015) for 13 WBs and for some samples from 11 WBs with pre- viously detected toxins (Tab. 3). The distribution of WBs with toxins (16), the WBs with recorded blooms (14), WBs in which toxic species have been found (30) and of the not problematic WBs (54) in the 8 groups of their geographic location and vertical position is shown on Fig. 7. According to the above cited references, 52 algae and some akinetes have been discovered in the water samples with detected toxins. Among them 45 from 21 genera were identified at species or generic level. The informa- tion on the number of their findings in toxic samples, the number of WBs, from which these samples have been col- lected together with the range of species biomass exactly in these samples, types of toxins, methods used and sources are summarized in Tab. 4. Figs. 2 and 3 illustrate the distribution of these species and genera in Bulgarian WBs during the studied period. Fig. 7. Distribution of water bodies (WB) with toxins, the WBs with recorded blooms, WBs in which toxic species have been found and of the not problematic WBs in the 8 phyla of their geographic location and vertical position (CV.I-LV.VI). Colours are on conformity with colours on Fig. 1, for the description of CV.I-LV.VI phyla see the text. No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 145 Ta b. 4 . C ya no pr ok ar yo te s fo un d in th e sa m pl es w ith d et ec te d to xi ns in B ul ga ri an W B s. Ta xo n N f N W B B io m as s M ic ro cy st in s R R Y R L A M C g O th er A N T- a S T X S ou rc ra ng ee (M C ) to xi ns L R N O D M ic ro cy st is a er ug in os a (K üt z. ) K üt z. 1 5 9 x- 1. 4 x x x x x x Pa vl ov a et a l. 20 06 , 2 01 5; T en ev a et a l. 20 09 , 2 01 0a . 2 01 1. 2 01 4; S to ya no v et a l. 20 12 A ph an iz om en on fo s- aq ua e R al fs e x B or n. e t F la h. 10 7 x- 2. 41 -D om x x x ? x x Pa vl ov a et a l. 20 06 ; T en ev a et a l. 20 09 , 2 01 0a , b , 2 01 4; S to ya no v et a l. 20 12 M ic ro cy st is w es en be rg ii (K om .) K om . I n K on dr at ie va 1 0 5 0 .2 -1 1. 97 * x x x P av lo va e t a l. 20 06 ,2 01 4, 2 01 5 P se ud an ab ae na c at en at a L au te rb . 6 1 x -0 .0 5 x x x x T en ev a et a l. 20 11 M ic ro cy st is fl os -a qu ae (W itt r.) K ir ch n. 6 5 0. 01 -7 .2 x x x P av lo va e t a l. 20 06 , 2 01 5; S to ya no v et a l. 20 12 Sy ne ch oc oc cu s el on ga tu s (N äg .) N äg . 6 3 0 .0 4- D om x x x x x T en ev a et a l. 20 10 a, b, 2 01 1 D ol ic ho sp er m um a ffi ne (L em m .) W ac kl in e t a l. 4 2 x -0 .0 3 x x x x x x T en ev a et a l. 20 09 ; 2 01 1 D ol ic ho sp er m um s ch er em et ie vi i ( E le nk .) w ac kl in e t a l. 4 1 0 .0 2- 0. 09 x x x T en ev a et a l. 20 11 A na ba en a sp . 4 4 0 .0 03 -8 .2 8 x x x P av lo va e t a l. 20 06 ; T en ev a et a l. 20 11 C hr oo co cc us m in ut us (K üt z. ) N äg . 4 2 0 .0 01 - x x x x x x T en ev a et a l. 20 10 b, 2 01 1 Sn ow el la la cu st ri s (C ho d. ) K om . e t H in d. 4 3 x -0 .0 2 x x x x ? x T en ev a et a l. 20 09 , 2 01 0a , b D ol ic ho sp er m um s pi ro id es (K le b. ) W ac kl in e t a l. 3 3 x- 6 5. 33 x x x 3 x T en ev a et a l. 20 10 b, 2 01 4; S to ya no v et a l. 20 12 M er is m op ed ia te nu is si m a L em m . 3 2 x -0 .0 2 x x x P av lo va e t a l. 20 14 . 2 01 5 M ic ro cy st is n at an s L em m . e x Sk uj a 3 3 0. 05 *- 1. 47 * x x x Pa vl ov a et a l. 20 06 M ic ro cy st is sp . 3 2 0. 1* -n ot in d. x P av lo va e t a l. 20 15 D ol ic ho sp er m um fl os -a gu ae (B ré b. e x B or n. e t F la h. ) W ac kl in e t a l. 2 1 x x x x ? x Te ne va e t a l. 20 10 b D ol ic ho sp er m um s ol ita ri um (K le b. ) W ac kl in e t a l. 2 1 0. 1- 0. 18 x T en ev a et a l. 20 11 Tr ic ho rm us v ar ia bi lis (K üt z. e x B or n. e t F la h. ) K om . e t A na gn . 2 1 x x x x T en ev a et a l. 20 11 P la nk to th ri x ag ar dh ii (G om .) A na gn . e t K om . 2 2 2 .3 2- 10 .5 x S to ya no v et a l. 20 12 P ho rm id iu m s p. 2 1 x x Te ne va e t a l. 20 11 Sp ir ul in a m aj or K üt z. e x G om . 2 2 x -0 .0 3 x x x T en ev a et a l. 20 11 ; P av lo va e t a l. 20 15 A ph an iz om en on s p. ju v. 1 1 0 .0 98 x Pa vl ov a et a l. 20 14 A ph an oc ap sa d el ic at is si m a W . e t G .S . W es t 1 1 0 .6 x x Pa vl ov a et a l. 20 15 A ph an oc ap sa g re vi lle i ( B er k. ) R ab en h. 1 1 0 .6 2* x P av lo va e t a l. 20 06 A ph an oc ap sa s pp . 1 1 0 .0 04 * x x G eo rg ie va e t a l. 20 15 C hr oo co cc us a ph an oc ap so id es S ku ja 1 1 x x P av lo va e t a l. 20 15 C hr oo co cc us d is pe rs us (K ei ss l.) L em m . 1 1 0. 02 T en ev a et a l. 20 11 C hr oo co cc us s p. 1 1 0 .0 01 x x x Pa vl ov a et a l. 20 14 C yl in dr os pe rm op si s ra ci bo rs ki i ( W oł .) Se en ay a et S ub ba R aj u 1 1 x x S to ya no v et a l. 20 12 Le pt ol yn gb ya fo ve ol ar um (R ab en h. e x G om .) A na gn . e t K om . 1 1 0. 01 1 x x x Pa vl ov a et a l. 20 14 Li m no ra ph is h ie ro no m us ii (L em m .) K om . e t a l. 1 1 x x x Te ne va e t a l. 20 11 Li m no th ri x sp . 1 1 4 9. 93 * x x x Pa vl ov a et a l. 20 06 M er is m op ed ia g la uc a (E hr .) K üt z. 1 1 x x x Pa vl ov a et a l. 20 15 M er is m op ed ia h ya lin a (E hr .) K üt z. 1 1 0 .0 08 x x x Pa vl ov a et a l. 20 14 To b e co nt in ue d on n ex t p ag e No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.146 DISCUSSION The analysis of data on phytoplankton species compo- sition revealed that, in spite of being quite heterogeneous, they are generally available for 111 from 115 purposively studied WBs included in Tab. 1, except concrete data for the reservoirs Stoychovtsi, Karaisen, Luzhenska Bara and Aleksandrovo. However, the occurrence of cyanoprokary- otes in the last reservoir was documented as “presence of toxic species” (Cheshmedjiev et al., 2010a; Fig. 1). In all samples proceeded by us, cyanoprokaryotes were not found in 11 alpine lakes (Bunderishko ezero 9, Musalensko ezero 3, Ezero Bliznaka, Ezero Bubreka, Ezero Okoto, Ezero Sulzata, Kremensko ezero 2, Marichino ezero 2, Marichino ezero 3, Popovo ezero 2 and Vlahinsko ezero 1) and in the occasionally sampled reservoirs Yarlovets and Zhernov. Therefore, we argue that 99 WBs contain different number and amount of cyanoprokaryotes. With the total of 210 taxa found, they represent only 3.8% of the algal biodiversity of the country estimated as ca. 5,500 taxa by Stoyneva (2014), but comprise 36.4% from all 576 cyanoprokaryotes (Stoyneva et al., 2016). Coccal cyanoprokaryotes were the richest group both in species (85) and genera (31), while the number of heterocytous species and genera was the lowest. This result is on general conformity with the shal- low and eu- to hypertrophic character of most studied WBs, in which, according to our unpublished results, nitrogen was not a limiting factor (N/P ranged between 7 and 110). Similar conclusion could be made according to the data in the publications by Teneva et al., 2010a; Belkinova et al., 2014; Pavlova et al., 2015; Stoyanov et al., 2016, etc. The highest taxonomic diversity, expressed as number of species and genera, was detected in two WBs: Blato Sre- barna (85/35) and Vaya (63/30) (Fig. 4). Due to their high importance for conservation of rare and threatened species of national, European and global significance (Michev and Stoyneva, 2007; Vassilev et al., 2013), these two shallow lakes have been intensively studied before and during the analyzed period, and always have been outlined for their rich algal diversity (Michev et al., 1998; Stoyneva, 1998a, 1998b, 2003, 2014, 2015; Georgiev, 2012; Dimitrova et al., 2014a, 2014b). However, the same authors outlined the negative trends in their development with enhanced eu- trophication, cyanoprokaryote blooms (incl. the rare equi- librium states dominated by cyanoprokaryotes) formed by potentially toxic species. The negative effects of increased eutrophication due to long-term cage fish farming were stressed also for the mountain reservoirs Dospat and Kardzhali, where among the newly appeared group of cyanoprokaryotes the harmful Aphanizomenon flos-aquae, Dolichospermum spiroides and Planktothrix rubescens (De Candolle ex Gomont) Anagnostidis et Komárek partici- pated in the dominant complexes (Dochin and Stoyneva, 2014, 2015; Dochin 2015).Ta b. 4 .C on tin ue d fr om p re vi ou s pa ge . Ta xo n N f N W B B io m as s M ic ro cy st in s R R Y R L A M C g O th er A N T- a S T X S ou rc ra ng ee (M C ) to xi ns L R N O D M ic ro cy st is b ot ry s Te il. 1 1 0 .6 x x x Pa vl ov a et a l. 20 15 M ic ro cy st is fi rm a (K üt z. ) S ch m id le 1 1 0 .0 4* P av lo va e t a l. 20 06 M ic ro cy st is p ul ve re a (W oo d) F or ti in D e To ni 1 1 d om x x x ? x Te ne va e t a l. 20 10 b O sc ill at or ia a nn ae V an G oo r 1 1 x x x Te ne va e t a l. 20 11 O sc ill at or ia s p. 1 1 0 .0 3 x Te ne va e t a l. 20 09 P la nk to th ri x co m pr es sa (Ü te rm oh l) A na gn . e t K om . 1 1 0 .0 7 x Te ne va e t a l. 20 11 P se ud an ab ae na li m ne tic a (L em m .) K om . 1 1 0 .1 x T en ev a et a l. 20 11 P se ud an ab ae na m uc ic ol a (N au m an n et H ub .-P es t.) B ou rr . 1 1 0. 11 8 x x x Pa vl ov a et a l. 20 14 R ap hi di op si s m ed ite rr an ea Sk uj a 1 1 0. 00 1- 0. 11 8 x P av lo va e t a l. 20 14 W or on ic hi ni a na eg el ia na (U ng .) E le nk . 1 1 0. 01 * x x x Pa vl ov a et a l. 20 06 W or on ic hi ni a sp . 1 1 0. 48 * x x x Pa vl ov a et a l. 20 06 U ni de nt if ie d [s ol ita ry c el ls . a ki ne te s. fi la m en ts -2 . c oc ca l c ol on ia l - 1] 9 6 x- 22 .5 4* x x x P av lo va e t a l. 20 06 , 2 01 4, 2 01 5 N f, nu m be r o f f in di ng s/ sa m pl es ; N W B , n um be r o f W B s in w hi ch th e sp ec ie s w as re co rd ed ; b io m as s ra ng e, re la te d to th e sp ec ie s in th e “t ox ic ” sa m pl es , M C g, in di ca te d on ly a s m ic ro cy st in s in th e pu bl ic at io ns (w ith ou t t he ir e xa ct ty pe s) ; x , p re se nc e; D om , d om in an t s pe ci es /d om in an ce ; * bi om as s es tim at ed fo r t hi s st ud y; n ot in d. , n ot in di ca te d in th e pu bl ic at io ns . No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 147 The analysis of the distribution of species and genera points to 2 species (Aphanizomenon flos-aquae and Cylin- drospermopsis raciborskii) and 11 genera as being the most widespread in the country (Planktothrix, Cylindrospermop- sis, Dolichospermum, Chroococcus, Microcystis, Pseudan- abaena, Anabaena, Aphanocapsa, Planktolyngbya and Aphanizomenon; Figs. 2 and 3). Except for Chroococcus, Aphanocapsa and Planktolyngbya they have been com- monly pointed as principally responsible for forming blooms (e.g. Oliver and Ganf, 2002) mainly due to presence of gas vesicles. Among the total of 69 genera found, at least 29 are known as cyanotoxin producers (Metcalf and Codd, 2012; Pettersson and Pozdnyakov 2013). If the genera Chrysosporum and Sphaerospermopsis, which relatively recently have been separated from Anabaena (Komárek, 2013 and references therein), are considered, this number will increase to 31, or 45% from all found in the country. The frequency of findings of all these potentially toxic gen- era in combination with the spreading of cyanoprokaryote blooms (Fig. 1) are logically related with the findings of different types and amounts of cyanotoxins in Bulgarian WBs (Tab. 2). The results of multivariate analysis show the consis- tency of the WBs classification based on geographic po- sition and altitude (as first steps proposed in the hierarchical classification of Bulgarian wetlands by Michev and Stoyneva, 2007), along with in-lake charac- teristics as depth and nutrient content. Total phytoplank- ton biomass, total cyanoprokaryote biomass, as well as cyanoprokaryote assemblages showed a strong response to the environmental variables, with an expected major influence of TP. The redundancy analysis identified four groups of genera in relation to WBs, among which groups 3 and 4 are more heterogenous in comparison with groups 1 and 2. Group 3 contains realtively small colonial embedded in mucilage coccal genera (Aphanocapsa, Chroooccus, Coelosphaerium, Snowella, Woronichinia) and non-colo- nial coccal Synechocystis which are classical phytoplank- ters in various types of WBs. Despite their general ubiquity and abundance in a wide spectrum of trophic conditions, these non-bloom formers are not a well-known group, particularly in relation to their ecology (Stockner et al., 2002). In our opinion, the presence of all these small coccal genera in group 3 reflects well the heterogenous character of its WBs in terms of geographic location, mor- phometry and way of use, but united by the prevalence of meso- to oligotrophic conditions. Within the group 4 with predominantly high conduc- tivity conditions, characterized by extremely low TBC, are associated diverse and most rarely (in this study) distrib- uted smallest coccal cyanoprokaryotes (all rare colonial non-toxic species, Merismopedia, non-colonial picoplank- ters of Pcy group), very thin filamentous rare Spirulina and Glaucospira species with a single (and most probably ben- thic) Phormidium. All of them are generally considered as non-blooming cyanoprokaryotes (Stockner et al., 2002). By contrast, most genera well known for their ability to form water blooms due to presence of gas vesicles (Oliver and Ganf, 2002) were in group 1: Microcystis; Anabaena s.l. (mainly Dolichospermum), Aphanizomenon s.l., Cylin- drospermopsis and Planktothrix. The other genera in group 1 are filamentous without gas vesicles: classical planktonic thin Planktolyngbya s.l., generally short-celled fine Rome- ria and occasionally distributed in this data set short-celled Oscillatoria and Borzia. As it could be seen, besides the very rare and therefore non-representative Osbr, the “ex- ception” from this filamentous group 1 is the coccal colo- nial Microcystis. However, the “filamentous” character of another participant in group 1 - Romeria - is still not proved and its coccal nature is in discussion (Komárek and Anagnostidis, 2005). Common feature of the strangers (Microcystis, Planktolyngbya and Romeria) in group 1 is the presence of well-developed homogenous colourless mucilage. The irregular presence of gas vesicles in com- bination with wide, but fine mucilage envelopes is known also for Anabaenopsis and Pseudanabaena, which are rep- resentatives of group 2. The group includes also the thin (1-2 µm) filamentous rarely branched Plectonema and straight filamentous Leptolyngbya representatives, most of which have not been determined at species level due to their small dimensions and problematic taxonomy (Komárek and Anagnostidis, 2005). The stranger in the group is the heterocytous and capable of bloom-forming Anabaenopsis. However, a capability for nitrogen fixation was proved for some Leptolyngbya species also (e.g. Stal, 2012). According to our recent knowledge, very few data confirm the toxic abilities of the genera in group 2 (except for Anabaenopsis milleri Voronichin, which was not found in our studies), which comprises coastal and inland low- land lakes and reservoirs with generally hypertrophic char- acter. By contrast, the genera from group 1, and its core representatives (Aphanizomenon, Cylindrospermopsis, Mi- crocystis and Planktothrix) in particular were repeatedly pointed as bloom-forming and toxin producing genera re- lated mainly with eutrophic conditions and were also the key players in toxic samples, detected in Bulgarian waters (Fig. 3; Tab. 2). They were found among the genera with broadest distribution in the country (Fig. 3). The common traits of the taxa belonging to this group are the affinity for eutrophic conditions and the presence of gas vesicles, which provide buoyancy control of filament and colonies, allowing efficient vertical migration in the water column of relatively shallow lakes which present alternation of pe- riods of stratification and mixing (Reynolds, 2006). Ac- cordingly, the WBs of group 1 are relatively small, inland lowland to low-mountain, eu- to hypertrophic reservoirs. The samples with detected cyanotoxins were from 16 No n- co mm er cia l u se on ly M.P. Stoyneva-Gärtner et al.148 WBs (Tab. 2) and contained 44 cyanoprokaryotes identi- fied to species or genus level (Tab. 4). Their distribution in Bulgaria, with a few exceptions, is relatively broad (Figs. 2 and 3). The results from this study confirm the trend for their fast spread in the country outlined espe- cially in relation to the invasive species like Cylindros- permopsis raciborskii (Stoyneva, 2015; Kokocinski et al., in press). Most of these 44 species and 21 genera have been repeatedly reported as real or potential toxin produc- ers but for some of them discussions are still running (e.g. for Microcystis species as summarized in Šejnohová and Maršálek, 2012). It is out of scope of this study to go deep in the contradictory opinions, which have to be interpreted with caution, moreover for details in relation with findings in Bulgaria we can refer to our previous papers (Pavlova et al., 2006, 2014, 2015). Summarizing the results from this study, we could outline that exactly one of the most often discussed species - M. wesenbergii - was the most often recorded species in toxic samples (Tab. 4). In the same time, Pseudanabaena mucicola (Naumann et Huber-Pestalozzi) Bourrelly, which is a relatively regular endophyte in M. wesenbergii, was reported only once as associated with cyanotoxin detection. Some of the species in Tab. 4 are identified only at genus level, or are known for problematic identification, and therefore discussion on them is not relevant without proper documentation, which is not available in the published papers. It has to be noted also, that other cyanoprokaryotes, known for their poten- tial to produce toxins, were found in the country. For ex- ample, Nodularia spumigena Mertens ex Bornet ex Flahault was reported from three temporary WBs on Be- lene island in The Danube (Beshkova and Botev, 2004), but the study was not supplied with toxin analysis. Obvi- ously, aimed studies on toxin groups other than micro- cystins, combined with permanent toxin monitoring will reveal more objective picture of presence/absence of dif- ferent cyanotoxins and species related with them. A co- occurrence of other algae could also have an effect, and even provide a source, for some of the toxins detected (e.g. pyrrhophytes for saxitoxins). Since long time it is well-known that cyanoprokaryote dominance (and blooms in particular) is strongly affected by complex in- teractions between lake morphometry, water temperature, underwater light availability, nutrient supply and total food-web structure and can not be viewed independently from all other members of phytoplankton (Harris, 1986; Reynolds, 1987, 2006; Dokulil and Teubner, 2000 among the many others). Therefore, we would like to outline the necessity for providing data in future publications on the total species composition for the samples and WBs with detected cyanotoxins or harmful blooms. In the temperate country like Bulgaria it is not surpris- ing that cyanotoxins were detected in the water samples from summer-autumn periods, when the abundance of cyanoprokaryotes is normally the highest for the year. However, the comparison of Tabs. 2 and 3 clearly shows that occurrence of toxins is intermittent in Bulgarian WBs. Conclusions about the correlations between finding of cyanotoxins and relevant species or total sample biomass are hardly possible due to lack of concrete published data for all cases. The only statement which could be done is that cyanotoxins were detected within a broad range of algal abundance (Tabs. 2 and 4). In some cases, Dolichos- permum spiroides (Klebahn) Wacklin et al., Limnothrix sp., Microcystis wesenbergii, Planktothrix agardhii (Gomont) Anagnostidis et Komárek, Anabaena sp. and some unidentified algae had high biomass values in sam- ples with detected toxins (Tab. 4). But toxins were de- tected even when species were found in low, even negligible, concentrations (the cases of Borovitsa, Val- chovets, Studena, etc. in Tab. 2). The topic was discussed by Pavlova et al. (2006) and Teneva et al. (2009), who supposed that such cyanotoxin quantities were due to blooms, which occurred in the WBs before the sampling. Therefore, it is necessary to outline again that finding of cyanotoxins in waters is not always related with obvious blooms. Moreover, the term bloom and its lower borders remained poorly defined (Oliver and Ganf, 2002). How- ever, the result obtained is important for rising the public awareness of the cyanoblooms, which could have long consecutive effect on the ecosystem health and humans. In spite of heterogenous way of representing the results on cyanotoxin amounts, it could be stated that in most cases, and in the drinking-water reservoirs in particular, the concentration of microcystin LR is lower than WHO’s (1998) limit of 1 μg L–1 (Tab. 2). The general amount and types of microcystins and other cyanotoxins are on con- formity with the results published for the closest neighbor- ing countries Macedonia, Serbia, Romania, Turkey and Greece, as it was outlined by Pavlova et al. (2015). How- ever, the fact of their findings in 16 WBs (among which 3 are important drinking-water reservoirs, 2 are water supply reservoirs, 3 are used for irrigation and 8 are recreational sites for sports and fishing) on the background of more recorded cyanoprokaryote blooms and broad spread of toxic species in Bulgaria (Figs. 1, 2 and 3) is strong enough to alarm both scientists and responsible authorities at na- tional level. Moreover, the analyses of all data clearly show that during the last 15 years cyanoprokaryotes were not re- stricted in distribution in the lowlands and plains but started to invade WBs with higher altitudes, i.e. WBs situated in kettles and low mountains (Fig. 7). The outlining of the ne- cessity of stronger recognition of the problem in the country and rising of the public awareness made here is not the first, just the opposite – almost each of the papers on the topic published in the analyzed period had this statement as a main conclusion since there is no doubt that the peculiar and otherwise fascinating group of cyanoprokaryotes is a No n- co mm er cia l u se on ly Cyanoprokaryote blooms and cyanotoxins in Bulgaria (2000-2015) 149 real hazardous factor for human and aquatic ecosystem health in Bulgaria. CONCLUSIONS All results from studies carried in summer- autumn periods of the last 15 years (2000-2015) in 120 Bulgarian WBs different in location, morphometry and trophic status (incl. drinking-water reservoirs, reservoirs for water sup- ply, irrigation and energy production reservoirs, recre- ational lakes and sites of nature conservation importance), gathered from 35 publications, showed that in 30 of them toxic species were found. Cyanoprokaryote blooms were recorded in 14 WBs and in 16 cyanotoxins (microcystins, nodularins and saxitoxins) were detected, and cyanoprokaryote diversity was quite high (210 taxa of 60 genera). Toxin concentration ranged between 0.1 and 26.5 µg L–1 in water samples and between 10.9 and 1070 µg g–1 (d.w.) in concentrated (net) samples. Despite the fact that microcystins were not found in all studied WBs and that the recorded levels were still lower in comparison with some other European countries and with WHO’s threshold for microcystin LR, the fact of cyanotoxin de- tection in 3 drinking-water reservoirs and cyanoprokary- ote occurrence in low mountain WBs could serve as an alert for the need of recognition of cyanotoxins as a new health risk factor in the country. Therefore, permanent monitoring with identification of toxins in WBs at risk and activities for limitation and control of toxic blooms are urgently needed, in combination with increase of the attention to the effects of cyanotoxins on both human health and health of aquatic ecosystems in Bulgaria. We also stress the need for a more comprehensive monitoring of the problematic WBs and of their watershed – includ- ing all key environmental variables (hydrology, nutrient loading and meteorology) and detailed phytoplankton sur- veys in order to improve water quality management and identify the measures to be taken to reduce the risks asso- ciated with cyanoprokaryote blooms. ACKNOWLEDGMENTS The authors would like to acknowledge the Euro- pean Cooperation in Science and Technology, COST Ac- tion ES 1105 “CYANOCOST- Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and man- agement” and Bulgarian national supporting financing by the Scientific Fund of the Ministry of Education and Sci- ence (projects DKOST 01/2-11.08.2016 and DKOST 10/2-19.08.2016) for adding value to this study through networking and knowledge sharing with European experts and researchers in the field. A part of the results, taken from Stoyneva (2014), was obtained during EMERGE project of EC Framework V, MEWs project on Srebarna Lake Monitoring (1994- 2003), project of World Bank and Zeleni Balkani for Man- agement Plan of Pomorie Wetland, monitoring studies of Bulgarian lakes of Ecotan EOOD commissioned by the EEA of MEWs of Bulgaria and studies on chosen olig- otrophic lakes in Pirin Mts, commissioned by Pirin Na- tional Park. 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