Nowadays, there is an active spread of cyanobacteria, primarily of the Nostocales order, in the surface waters of Europe and an increase in the number of cases of water blooms with their involvement (Taranu et al. 2015). As recently shown, the Nostocales are expected to expand into new habitats and then integrate into ecosystems, thus disrupting their resilience. This is proven, for example, by the increase in the prevalence of Raphidiopsis raciborskii in recent decades, especially in temperate climates (Rzymski et al. 2018). This phenomenon can be explained by high phenotypic plasticity, climate change and anthropogenic eutrophication (Cirés and Ballot 2016). It is obvious that freshwater reservoirs with a steadily elevated temperature regime provide a favorable environment for the expansion of toxic species of cyanobacteria to new areas with their subsequent spread.
Based on the data that the expansion of potentially toxic cyanobacteria began in latitudes with tropical and subtropical climates, we chose for the study reservoirs characterized by elevated temperatures due to geographical location (Kasperivtsi Reservoir and the Seret River below its dam) or operating conditions (Netishyn Reservoir). Phytoplankton analysis showed that cyanobacteria were the dominant fraction in all cases with a more visible predominance in summer . In particular, several potential producers of potent toxins were identified: Raphidiopsisraciborskii, Aphanizomenongracile, Dolichospermumflos-aquae. However, screenings for dissolved or partial cylindrospermopsin, microcystins (-LR, -YR, and -RR), and toxoid were negative.
The cyto- and genotoxic properties of cylindrospermopsin, an alkaloid biosynthesized by certain species of cyanobacteria, have been extensively studied in experimental in vitro and in vivo models. Several independent studies have separately identified the role of uracil, guanidine, and the hydroxyl group in CYN toxicity (Evans and Murphy 2011; Cartmell et al. 2017). In the proposed study on a model of normal carp hepatocytes in vitro, the toxicity of five synthetic simplified analogues was tested, all of which contained a uracil component in their structure, but differed in combinations of other components and functional groups inherent in CYN. All tested analogues caused the reactive oxygen species, lipid peroxidation and increased levels of DNA fragmentation/ The greatest increase in TBARS content and activity of caspase-3 (a marker of apoptosis) was demonstrated by an analogue that, like CYN, contains functional groups of guanidine, hydroxyl and uracil, but does not have its complex tricyclic structure. Observations made in the course of this work confirm the hypothesis that the toxicity of CYN is the result of the interaction of the components of uracil, guanidine and the hydroxyl group. Certain species of cyanobacteria and green algae are known to produce lipophilic polymethoxy-1-alkenes, which can cause teratogenicity invivo. Due to the insufficient level of information on PMA in Arthospira sp. (commercially known as spirulina) and Chlorella sp., which are cultivated for the production of food additives, we analyzed the level of biosafety of food additives Chlorella (n = 10) and Spirulina (n = 13) registered in the EU. Mass spectrometric analysis of the fractionated extracts did not reveal any potentially related chemical compounds in the studied materials. The analyzed samples did not show teratogenic effects, except for curvature of the spine caused by fractions of two chlorella drugs. However, the analyzed supplements caused oxidative stress and cytotoxicity in zebrafish liver tissue, as indicated by increased levels of reactive oxygen species, catalase activity, lipid peroxidation and increased levels of DNA fragmentation. The majority (60%) of chlorella fractions caused cholinesterase activation in zebrafish brain, while the effect of 61.5% of spirulina fractions caused the opposite reaction - its suppression. The effects of lipophilic extracts, especially Chlorella, on Danio rerio were similar to the response to cyanobacterial extracts, which may indicate the similarity of toxic metabolites produced by both groups or contamination of bioadditives with blue-green algae. Application to the studied samples of the method of detection of potentially toxic blue- green algae developed on the basis of the studies described above (Falfushynska and Horyn, 2019) showed that for Ch5,7 and Sp 12 the level of biosafety corresponds to the indicator “stress” (acute stress response), and for Ch6 and Sp2, 6 - “pre-stress state” (adaptive response to damage).
Therefore, although our studies confirm the absence of teratogenic compounds belonging to the class of polymethoxy-1-alkenes in the Chlorella and Spirulina preparations, their cytotoxic effect attests the necessity of further research.
Key words: cyanobacteria, cyanotoxins, carp fish, biomarkers, oxidative stress, cytotoxicity, genotoxicity, neurotoxicity.
