Cyanoprokaryotes (=cyanobacteria = blue-green algae) are photosynthetic prokaryotic organisms which have inhabited the Earth for approximately 3.5 billion years [1] . Recently they continue to occur naturally in different surface waters. Under some favourable conditions (e.g. nutrient enrichment, increased temperature, pH, water column stability, and insolation) they can rapidly reproduce and multiply cause “blooms” [2] -[5] . These blooms can be harmful to the environment, animals, and human health since some species of cyanobacteria produce toxic secondary metabolites, known as cyanotoxins ( [6] [7] among the many others). The most widespread cyanotoxins are the cyclic peptide toxins called microcystins (MCs), from which at least 80 are known, including microcystin-LR (MC-LR) [8] . The last is generally considered one of the most toxic [8] and therefore World Health Organization (WHO) has recommended a provisional guideline value for MC-LR 1 µg/l for drinking water [9] . Until nowadays most of the developed regulations or guidelines for MCs in drinking water and recreational waters in different countries of the world are based on this value [8] . In spite of the recognition of the problem of water blooms, and real and potential presence of MCs in Bulgarian waters, the studies on the topic are scarce ([10] and citations there) and no regulatory guidelines for cyanotoxin content in drinking and recreational waters exist in the Bulgarian legislation. This study presented the results of MC analyses by HPLC DAD from five Bulgarian water bodies (reservoirs Stoudena, Pchelina and Bistritsa, and lakes Dourankoulak and Vaya) carried out in 2012-2014, as a continuation of our previous studies [10] - [13] ; and first data on MCs in the lake MominBrod were detected in the period 2012-2014.

During the study, MC-LA and nodularin were not found, but presence of other MCs (-LR, -YR, -RR) was proved for all investigated water bodies (except Lake Vaya) in the period 2013-2014, while in 2012 MCs were detected in measurable amounts only in the samples from the lake Momin Brod. The cyanoprokaryotic taxa found and their biomass, the MC concentrations together with the amounts of total nitrogen and phosphorous and the N/P ratio are shown in Table 1.

The occurrence of MCs in the examined water bodies was irregular in terms of time (Table 1). When found, the concentrations of intra + extracellular MCs in water samples ranged from 0.37 to 1.8 µg/l, while the amount of total MCs in the net samples ranged from 10.9 to 296.1 µg/g (d.w.) (Table 1).

In the samples from the lake Vaya, collected in 2012 and 2013, MCs were not detected. The same was the situation in 2011 and 2005 and only earlier, in 2004 -RR, -YR and -LR were found there [10] - [13] . The lack of MCs is explainable by the species composition in the lake at the time of sampling in this (Table 1) and previous [24] - [28] studies. However, in all samples some potential producers of other cyanotoxins were recorded. For example, the neurotoxin anatoxin-a (ANA) “has been detected in natural populations of cyanobacteria when Anabaena, Oscillatoria, Aphanizomenon, Cylindrospermum, Planktothrix, and Raphidiopsis dominate the cyano- bacterial community” [29] . ANA-producing strains have been isolated from all of these genera [30] [31] and representatives of all them (incl. Dolichospermum as recently splitted from Anabaena [17] [20] ) have been found in significant amounts in the lake waters in different periods (Table 1, [14] [24] - [28] ). Concerning biomass, Planktothrix agardhii is one if the “key players” in the phytoplankton of Lake Vaya [14] [25] - [28] . For this species toxicity and presence of high amounts of MCs was indicated [e.g. [32] and in addition toxic effects independent of MC content were proved [33] . From the potentially toxic (and frequently reported as toxic [32] , capable also of cylindorspermopsin production [34] [35] ) species Aphanizomenon flos-aquaethe variety klebahnii was separated. The last seems to be more widespread than the typical variety and most recently it was re- cognized as a separate species [17] [20] . A. klebahnii was also found in the phytoplankton of Vaya Lake, and in spite that it is more often regarded as non-toxic [32] its presence still has to be considered with carefulness when harmful blooms are discussed. The high values of Cylindrospermopsis raciborskii biomass, detected in 2013 (Table 1), confirm the relatively stable “lodging” there of this exotic species, found in big amounts in the lake since 2000 [25] - [28] . C. Raciborskii is well known for producing toxins, including cylindrospermopsin (hepato- toxin; CYN) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed (e.g. [31] [35] - [38] ). In addition to the dominant toxin, the toxic alkalloid CYN, recently a deoxy-cylindrospermopsin was is- olated from Japanese strain ISG9 of this species [39] and some other toxic metabolities were found in a strain from a temperate zone, unable to synthesize CYN [40] . This first study on the cell-free extracts of a non-CYN- producing strain of C. raciborskii in human cells (neutrophils and lymphocytes) showed that the observed effects were compared to those induced by CYN (1.0 - 0.01 μg∙mL⁻¹) and also some other effects (e.g. increased necrosis and apoptosis in neutrophils and elevated apoptosis in lymphocytes), not induced by CYN, regardless of the toxin concentration, were registered. From these results was concluded that strains occurring in temperate zones may pose a threat to human health through the production of hitherto unknown metabolites that reveal a toxic pattern different to that of CYN [40] . This discovery again shows the possibility of C. raciborskii to cause water-safety problems and emphasizes the need for basic monitoring schemes for this species in water supplies, especially in those located in the temperate regions. Therefore, in spite of the recent negative results, the high MC concentration in 2004 [11] - [13] together with the presence of potential producers of other cyanotoxins and toxic metabolites on the background of the strong blue-green algal blooms, observed since years in the lake ( [14] [24] - [28] Table 1) suggest the necessity of permanent monitoring of Vaya and prove its inclusion in the Red List of Bulgarian wetlands in Critically Endangered category [41] [42] .

During this study, MCs were not detected also in the reservoir Bistritsa, with one exception of the sample examined from water remains after the draining of the reservoir in 2013 (Table 1). The same was the situation in 2011 and in 2005, when MCs were not found. Only once, in 2004, extracellular MCs were identified in reservoir waters [10] [12] [13] . In all samples, which show positive results for MC presence in this and previous studies, the microscopic work revealed presence of Microcystis species (Table 1, [10] ). The lack of MCs in most of the samples is easily explainable with the phytoplankton species composition, in which different, but non-toxic representatives from almost all algal groups have been determined (e.g. from genera Ceratium, Euglena, Pediastrum, Phacotus, Phacus, Polyedriopsis, Tetraedron, Trachelomonas, among the many others)

Results from the sample analyses for the mountain drinking water reservoir Stoudena show general lack of MCs, with the exception of both quantitative water and net samples from October 2013 (Table 1). The same was the situation with our previous studies of this reservoir, when only once, in 2011, 0.1 mg/l MYC-LR was found [10] , while in all samples from 2004 and 2005 no MC was detected [11] - [13] . The amount of MC in net samples from the same date of October 2013, as it could be expected, is much higher (10.9 mg/g in total, and 8.11 mg/g of LR) than in water samples (only 01 mg/l MYC-LR) (Table 1). During this study, for first time -YR and -RR were found in the examined samples. This, together with the confirmation of the presence of MYC-LR ―one of the most toxic cyanotoxins―in this drinking water reservoir, confirms our previous opinion and proposal [10] for stronger permanent control of its water quality and necessity of taking measures with appropriate steps for its improvement in the nearest future. In spite of the fact that the concentration of the toxin is 10 times lower than WHO limit of 1 µg/l, the data obtained on both higher MC presence and more abundant development of cyanoprokaryotic taxa (which were not typical for this water body [14] ) are a strong alert about on-going negative processes in this reservoir. They are probably due to a combination of factors leading to a general worsening of the sanitary protection of the water basin (contamination with nitrogen and phosphorous, data about which at present are unavailable [10] ) on the background of global warming, which seems to enhance the development of cyanoprokaryotes.

During this study MCs were found in Dourankoulak Lake only in the samples from 2013 (Table 1). Their concentrations are much lower in water samples when comparison with our previous results on MCs from 2004, 2005 and 2011, obtained by HPLC and DAD is made [10] - [13] . The algological studies show permanent pre- sence of potentially toxic cyanobacteria in this coastal wetland during the last 22 years [11] [25] [28] [43] [44] . Nevertheless that in this studythe concentration of MYC-LR in the water sample is lower than maximal per- missible level 20 µg/l for bathing water according to [45] in the country (as was the situation in 2011 [10] ), we confirm our previous opinion that this lake, undergoing strong eutrophication process, should be object of special monitoring, restoration measures and conservation activities [10] [11] [43] [44] , as well as its inclusion in the Red List of Bulgarian wetlands in Critically Endangered category [41] [42] . The contradictory opinions on the toxicity/non-toxicity of one of the most often found and important species in the phytoplankton of Douran- koulak―Microcystis wesenbergii―were discussed in our previous paper [10] . Here we would like to add that in 2012 it was indicated again as a “definitely toxigenic species” [46] and included in the “list of species that have been confirmed to have toxin-producing strains” [47] . Two more Microcystis species―Microcystis flos-aquae and M. viridis―were identified in MC-containing samples from the lake (Table 1). The both species were noted among the potentially toxic producers ( [32] and citations there). However, in comparison with M. viridis, for which toxicity and especially MC presence were often indicated (e.g. [6] [48] - [52] and citations therein), according to our knowledge, M. flos-aquae was much more rarely definitelypointed as a toxigenic species. For example,its toxic and lethal effects on animals were noted [53] [54] and later a study of its strain from a Spanish-drinking water reservoir Santillana proved that it contained 4 major MCs: MC-LR, MC-LF, MC-LW, and MC-LY [55] .

Positive results were obtained from one sampling also in the reservoir Pchelina, where in October 2014 three MCs (-YR, -RR and -LR) in both quantitative water and net samples were found (Table 1). The microscopic investigation revealed only presence of big amount of separate, identical ellipsoidal cyanoprokaryotic resting cells―akinetes, the identification of which is impossible on species level. Their occurrence clearly shows that left behind was a development of heterocystous filamentous algae. Such types of akinetes are known for example in genera Anabaena L., Anabaenopsis, Aphanizomenon, etc. [17] [18] , some species of which are capable of cyanotoxin production [6] [7] . In the samples from 2011, when only MC-RR was detected, Aphanizomenon sp. was found in the summer-late summer samples of the same reservoir [10] . By contrast, in 2012, when a bloom of Anabaena sp. st. was found, no MCs were detected in water and net samples (Table 1). In our previous studies of the same reservoir in 2004 all three MCs were found in the “biomass” from net samples (LR + RR + YR/LR = 536/140), but dissolved MC were not found in water samples [12] . The confirmation of MC presence and their high concentration in net samples are relevant to our previous statement that in certain conditions this reservoir may be hazardous for a recreational use [10] .

The results of N/P ratio analysis show its high value (ranging between 17 and 218―Table 1) and indicate the P limitation of all studied water bodies according to [23] . However, due to low amount of samples investigated, no significant correlation was found between MC presence, quantities and amounts of these biogenes.

The detected maximum concentrations of MCs from Bulgarian water bodies are lower in quantitative water samples, but higher in net samples, when a comparison with our previous study [10] is made. As discussed there, they are still close to the quantities reported from other European countries (e.g. [55] - [62] ) and are still lower in comparison with the amounts found in the Czech Republic―up to 36 µg/l dissolved MCs in water bodies [63] and Slovakia―up to 1445.5 μg/g d.w. in “biomasses” [64] . The values obtained by us during this and previous studies [10] - [13] are generally higher in comparison with other investigations, revealing MCs in Bulgaria (e.g. [65] - [67] ). This result is explainable mostly with the differences in the geographic location, morphometric features and detected biodiversity in the examined water bodies [14] .

The results from this study, obtained during the period 2012-2014, show the presence of cyanotoxins MCs in five of the six investigated water bodies. When found, they ranged from 0.1 to 1.8 µg/l in quantitative water samples and from 1.35 to 296 µg/g d.w. in concentrated biomasses from net samples. Despite the fact that in 2012 MCs were not found in five of the all six studied water bodies and that the registered MCs were still lower in comparison with some other European countries, the presence of samples in which the most toxic MC-LR was recorded could serve as a strong alarm for the necessity of a serious study and relevant discussion of the problem with responsible authorities at national level with need of recognition of the problem as a new health risk factor. This statement conforms to our previous conclusions [10] and finds additional support in the recorded high biomass of cyanoprokaryotes, which indicates presence of their blooms in the studied wetlands. Considering that a lack of correlation between the toxicity and the MC content in cyanobacterial samples has also been observed [33] [68] , the presence of such blooms of potentially toxic species can serve as indication of potential risk and water-safety problems. Therefore, permanent monitoring with identification of toxins in water bodies 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.

Authors are thankful for the financial support of MESY scientific projects DDVU-02/77 and DFNI-T01/5 NANOSORBLAB. One of the authors of this paper, B. Uzunov, participates in the Project № BG051PO001- 3.3.06-0045/18.07.2013 in Human Resources Development Operational Programme, to which thankfulness is due. The authors would like to acknowledge the European Cooperation in Science and Technology, COST Action ES 1105 “CYANOCOST―Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management” for adding value to this study through networking and knowledge sharing with European experts and researchers in the field.

VeraPavlova,MayaStoyneva-Gärtner,BlagoyUzunov,BlagoyUzunov,ZlatkaBratanova,AntoanetaLazarova,IrinaKaradjova, (2015) Microcystins-LR, -YR and -RR in Six Bulgarian Water Bodies of Health and Conservational Importance (2012-2014). Journal of Water Resource and Protection,07,1375-1386. doi: 10.4236/jwarp.2015.716111