Comparative evaluation of neonicotinoids and their metabolites-induced oxidative stress in carp primary leukocytes and CLC cells.

Neonicotinoids (NEOs) have been designed to act selectively on insect nicotinic acetylcholine receptors (nAChRs). However, nAChRs are also expressed in vertebrate immune cells, so NEOs may interfere with the immune system in exposed non-target animals. The present study shows that NEOs: imidacloprid and thiacloprid, and their main metabolites: desnitro-imidacloprid and thiacloprid amide, at sub-micromolar concentrations ranging from 2.25 to 20 µM, affect the immune cells of fish. This was found both in primary cultures of leukocytes isolated from the carp head kidney and in the continuous adherent carp monocyte/macrophage cell line. Moreover, the results revealed that the studied pesticides and metabolites generate oxidative stress in carp immune cells and that this is one of the most important mechanisms of neonicotinoid immunotoxicity. Significant increases were observed in the formation of ROS and malondialdehyde (MDA). The antioxidant status alteration was linked with decrease in antioxidant enzyme activity: superoxide dismutase (SOD), catalase (CAT), and non-enzymatic antioxidant glutathione (GSH). Importantly, the metabolites: desnitro-imidacloprid and thiacloprid amide showed significantly higher cytotoxicity towards fish leukocytes than their parent compounds, imidacloprid and thiacloprid, which emphasizes the importance of including intermediate metabolites in toxicology studies.

Studies on the Effects of Fermentation on the Phenolic Profile and Biological Activity of Three Cultivars of Kale.

Fermentation is used not only to preserve food but also to enhance its beneficial effects on human health and achieve functional foods. This study aimed to investigate how different treatments (spontaneous fermentation or fermentation with the use of starter culture) affect phenolic content, antioxidant potential, and cholinesterase inhibitory activity in different kale cultivars: 'Halbhoner Grüner Krauser', 'Scarlet', and 'Nero di Toscana'. Chosen samples were further tested for their protective potential against the Caco-2 cell line. HPLC-MS analysis revealed that the fermentation affected the composition of polyphenolic compounds, leading to an increase in the content of rutin, kaempferol, sinapinic, and protocatechuic acids. In general, kale cultivars demonstrated various antioxidant activities, and fermentation led to an increase in total phenolic content and antioxidant activity. Fermentation boosted anti-cholinesterase activity most profoundly in 'Nero di Toscana'. Extracts of spontaneously fermented 'Scarlet' (SS) and 'Nero di Toscana' (NTS) showed cytoprotective properties, as revealed by the malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) assays. Additionally, strong anti-inflammatory activity of NTS was shown by decreased release of cytokines IL-1ß and TNF-α. Collectively, the conducted studies suggest fermented kale cultivars as a potential source for functional foods.

Silver and Copper Complexes with Ibuprofen and Caffeine-Preparation and Evaluation of Their Selected Biological Effects.

Several organometallic complexes based on more than twenty different metals have already been approved for medical applications. The aim of the presented research was to obtain complexes of silver and copper with the non-steroidal anti-inflammatory drugs ibuprofen and xanthine alkaloid caffeine and evaluate selected aspects of their bioactivity and biosafety in terms of their future possible applications. The obtained complexes were characterized by Fourier-transform infrared spectroscopy, thermogravimetry, UV-VIS spectroscopy, conductometry, elemental analysis, and bioassays. Cytotoxicity for normal human cells of the CCD-Co18 cell line was evaluated by determining the IC50 value, with metabolic and morphology assessments. It was observed that complexes containing ibuprofen and caffeine exhibited lower toxicity than those with ibuprofen only. Complexes with copper showed lower toxicity towards healthy human fibroblasts compared to silver-based compounds, with an IC50 above 140 µg mL-1. However, in the silver complexes, the presence of caffeine increased the potency of COX-2 inhibition. Antimicrobial effects against different Gram-positive and Gram-negative bacterial strains were evaluated by MIC determination with values less than 20 µg mL-1.

Combined effects of two phytoalexins, brassinin and camalexin, on the cells of colorectal origin.

Brassinin and camalexin belong to phytoalexins, plant compounds generated in the response to stress. Both compounds are known to be cytotoxic to several cancer cell lines, mainly by inducing oxidative stress and subsequent apoptosis. In the presented study, cytotoxic effects of brassinin and camalexin, individually and, for the first time, after combined exposure, on the cells of normal (CCD-Co18) and cancer (Caco-2) lines originated from colorectal tissues and their proapoptotic impact on Caco-2 cells were studied. The determined IC50 values indicate a clearly higher sensitivity of cancer cells to the tested substances, as well as a stronger cytotoxic effect of camalexin than brassinin. The synergistic effect of both phytoalexins was also demonstrated. Caspase-dependent and independent mechanisms were involved in the final effects. Both tested phytoalexins caused evident, concentration-dependent symptoms of oxidative stress in cancer cells, leading to apoptosis, but in the highest concentrations, also to necrosis. In case of camalexin, signs of pyroptosis were additionally detected.

Mesoporous silica nanoparticles containing copper or silver synthesized with a new metal source: Determination of their structure parameters and cytotoxic and irritating effects.

One of the potential implementation of mesoporous silica nanomaterials (MSNs) is their use in biomedical applications as adsorbents or carriers of various bioactive substances. In this study, we attempted to fabricate silica nanomaterials containing copper and silver that were introduced into the MSN matrix, for the first time using oxalate compounds as a metal source. The syntheses were carried out using hydrothermal and impregnation methods. Structure studies revealed that the obtained nanoparticles were of a spheroidal shape and most had diameters in the range 200-500 nm. Silver and copper were found to be grouped into clusters in most samples, except in copper-decorated MSNs prepared with the impregnation method, which had an even distribution of metal atoms throughout the volume of the granule. An evaluation of the cytotoxic and irritating effects revealed that the preferred candidates for potential future applications in medicine or cosmetology among materials obtained with the presented method are the copper-conjugated MSNs.

Cytotoxic and immunological responses of fish leukocytes to nodularin exposure in vitro.

Nodularin (NOD) is a cyclic peptide released by bloom-forming toxic cyanobacteria Nodularia spumigena commonly occurring in brackish waters throughout the world. Although its hepatotoxic effects are well known, other negative effects of NOD have not yet been completely elucidated. The present study aims were to evaluate and compare the cytotoxic and immunotoxic effects of the toxin on primary leukocytes (from head kidney [HK]) and stable fish leukocytes (carp leucocyte cell line [CLC] cells). The cells were incubated with the cyanotoxin at concentrations of 0.001, 0.01, 0.05, or 0.1 µg/ml. After 24 h of exposure, the concentrations ≥0.05 µg/ml of toxin resulted in cytotoxicity in the primary cells, while in CLC cells, the toxic effect was obtained only with the highest concentration. Similarly, depending on the concentration, exposure to NOD causes a significant inhibition of chemotaxis of the phagocytic abilities of primary leukocytes and a significant reduction in the proliferation of lymphocytes isolated from the HKs. Moreover, CLC cells and HK leukocytes incubated with this toxin at all the mentioned concentrations showed an increased production of reactive oxygen and nitrogen species. NOD also evidently influenced the expression of genes of cytokine TNF-α and IL-10 and, to a minor extent, IL-1ß and TGF-ß. Notably, the observed changes in the mRNA levels of cytokines in NOD-exposed cells were evident, but not clearly dose-dependent. Interestingly, NOD did not affect the production and release of IL-1ß of the CLC cells. This study provides evidence that NOD may exert cytotoxicity and immune-toxicity effects depending on cell type and toxin concentration.

Assessment of the cytotoxic impact of cyanotoxin beta-N-methylamino-L-alanine on a fish immune cell line.

Beta-N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by several cyanobacteria species. It is considered to be a potent neurotoxin. Although its neurotoxic effects are well studied, other negative effects of BMAA have not yet been completely elucidated. In the present study, we studied the cytotoxic effects of a wide range of concentrations of BMAA (0.25-2.0 mM) on a stable fish immune cell line (CLC) obtained from carp monocytes. The cells exposed to higher concentrations of BMAA exhibited an altered morphology, changed ATP levels, and reduced proliferation. On the basis of toxic effects of BMAA on lysosomes, mitochondrial dehydrogenases activity, and cell membrane integrity, we determined its cytotoxic concentrations. We also investigated effects of the toxin at non-cytotoxic concentrations on the basic functions of CLC cells. BMAA did not affect the production and release of IL-1ß or phagocytic activity of the cells. However, higher non-toxic BMAA concentrations altered the levels of extracellular and intracellular total proteins compared to those in control cells.

A representative of arylcyanomethylenequinone oximes effectively inhibits growth and formation of hyphae in Candida albicans and influences the activity of protein kinases in vitro.

In this study, we applied various assays to reveal new activities of phenylcyanomethylenequinone oxime-4-(hydroxyimino) cyclohexa-2,5-dien-1-ylidene](phenyl)ethanenitrile (4-AN) for potential anti-microbial applications. These assays demonstrated (a) the antimicrobial effect on bacterial and fungal cultures, (b) the effect on the in vitro activity of the kinase CK2, (c) toxicity towards human erythrocytes, the Caco-2 cancer cell line, and embryonic development of Zebrafish. We demonstrated the activity of 4-AN against selected bacteria and Candida spp. The MIC ranging from 4 µg/ml to 125 µg/ml proved effective in inhibition of formation of hyphae and cell aggregation in Candida, which was demonstrated at the cytological level. Noteworthy, 4-AN was found to inhibit the CK2 kinase with moderate potency. Moreover, at low concentrations, it did not exert any evident toxic effects on human erythrocytes, Caco-2 cells, or Zebrafish embryos. 4-AN can be a potential candidate as a novel drug against Candida infections.

Comparative studies on the cytotoxic effects induced by nodularin in primary carp leukocytes and the cells of the fish CLC line.

Nodularin, one of the toxins produced by cyanobacteria commonly occurring in brackish waters, is a cyclic pentapeptide, that may exert adverse effects on animal and human health. The aim of the present study was to evaluate the cytotoxicity of nodularin on different fish cellular models. The leukocytes isolated from carp head kidney and common carp (Cyprinus carpio L.) leukocyte cell line (CLC) were incubated with the cyanotoxin at the concentrations of 0.001, 0.01, 0.05 or 0.1 µg/mL. Treatment of cells with the higher concentrations of the toxin resulted in a dramatic decrease in cell viability. Simultaneously, we observed an increase in the percentage of necrotic cells. Nodularin also induced apoptosis as demonstrated by increased caspase-3/7 activity, and DNA fragmentation. We found, that primary cell cultures were more sensitive to the toxin than CLC cells. Our study also demonstrated significant increases in intracellular ROS production and decreases in GSH levels, observed in both primary leukocytes and CLC cells exposed to nodularin at 0.01-0.1 µg/mL. These cells exhibited similar GSH/GSSG ratios and similar level of oxidized glutathione.

Effects of cylindrospermopsin on the phagocytic cells of the common carp (Cyprinus carpio L.).

Cylindrospermopsin is a cyanotoxin with cytotoxic activity. It is released into water during and after cyanobacterial water blooms and thus poses a threat to the health of fish. There is very little information available concerning the effects of the toxin on fish immune cells. In this study, we assessed the potential impact of cylindrospermopsin on the basic functions of phagocytic cells from common carp (Cyprinus carpio L.), including phagocytosis, reactive oxygen and nitrogen species production, and the structure of microfilaments and selected cytokine expression. Phagocytic cells, isolated from fish head kidneys, were exposed to the toxin at concentrations of 0.05, 0.1, 0.5 or 1 µg ml(-1), for up to 24 h. Cytotoxicity, detected by lactate dehydrogenase release, was observed at the highest studied concentration. A decrease in phagocytic activity and changes in actin cytoskeletal structures were observed after the cell exposure to the toxin at 0.5 and 1 µg ml(-1). Moreover, at all tested concentrations, cylindrospermopsin increased the production of reactive oxygen and nitrogen species. It also evidently influenced the expression of genes of proinflammatory cytokines interleukin-1ß and tumour necrosis factor-α and, to a minor extent, anti-inflammatory transforming growth factor-ß, but had no effects on interleukin-10. The results indicated that the cyanotoxin cylindrospermopsin is able to modify basic features of carp phagocytic cells, which might result in adverse consequences for fish health.

Cylindrospermopsin induces oxidative stress and genotoxic effects in the fish CLC cell line.

Cylindrospermopsin (CYN) is a cyanotoxin detected in water reservoirs worldwide. The toxin is a potent protein synthesis inhibitor capable of adversely influencing a wide range of cell functions. While data on the prooxidative potency of CYN are inconsistent, genotoxic effects towards certain mammalian cell types have been described. However, such a potential on fish cells has not yet been investigated. Hence, the aim of the study was to elucidate the prooxidative and genotoxic impact of CYN on a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 0.1, 0.5 or 1 µg ml⁻¹. After 24 h, cytotoxic activity of CYN at the highest used concentration was confirmed by decreased cell membrane integrity and inhibited cell proliferation. Additionally, CYN at 0.5 and 1 µg ml⁻¹ increased intracellular ATP levels and decreased the reduced to oxidized glutathione ratio. Furthermore, a significant increase in the reactive oxygen species (ROS) production with concomitant changes in superoxide dismutase activity was observed after a 3.5-h exposure of the cells to the toxin. Genotoxic activity of CYN, manifested as oxidative DNA damage and elevated number of micronuclei, was also detected in exposed cells. The obtained results indicate that CYN is able to exert a wide range of adverse effects, including oxidative stress and genotoxicity in fish leucocytes.

Effects of cylindrospermopsin on a common carp leucocyte cell line.

Cylindrospermopsin (CYN) is a cytotoxin produced by different cyanobacterial species, increasingly detected in water reservoirs worldwide. There is very little information available concerning the effects of the toxin on fish immune cells. The aim of the study was to elucidate the potential impact of cylindrospermopsin on the selected parameters of a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 10, 1 or 0.1 µg ml(-1) for up to 48 h. Cell viability and proliferation, apoptosis/necrosis induction, cell morphology and phagocytic activity were determined. The two higher toxin concentrations occurred to be evidently cytotoxic in a time-dependent manner and influenced all studied parameters. The lowest used concentration had no effects on cell viability and cell number; however, a strong reduction of bacteria uptake after 24-h exposure was detected. The obtained results indicate that cylindrospermopsin may interfere with the basic functions of fish phagocytic cells and as a consequence influence the fish immunity.

Cytotoxic effects and changes in cytokine gene expression induced by microcystin-containing extract in fish immune cells--an in vitro and in vivo study.

Blooms of cyanobacteria producing very toxic secondary metabolites (especially microcystins) are potent environmental stressors, hazardous not only to aquatic animals but also to public health. The purpose of this study was to investigate the effects of an extract containing microcystins on immune cells isolated from the common carp (Cyprinus carpio L.). In the present study it has been found that the extract induced apoptosis and inhibited in vitro lymphocyte proliferation. In addition, the results indicated the possible role of oxidative stress in this cytotoxicity and apoptosis. The in vivo investigations showed that the extract containing microcystins had greater suppressive effects on the essential functions of immune cells (intracellular reactive oxygen species production and lymphocyte proliferation) than the pure toxin alone. Moreover, immersion of fish in the toxic extract caused changes in the mRNA levels of various pro- and anti-inflammatory cytokines in carp leukocytes, while after exposure to the pure toxin, only IL1-ß expression was markedly up-regulated. The observed modulatory effects on immune cells could have important implications for the health of planktivorous fish, which feed more frequently on toxic cyanobacteria.

Microcystin-LR induces cytotoxicity and affects carp immune cells by impairment of their phagocytosis and the organization of the cytoskeleton.

Microcystin-LR (MC-LR) is the main isoform of hepatotoxin produced by cyanobacteria, which occur worldwide in the aquatic environment. The present study investigated the in vitro toxic MC-LR effects on immune cells isolated from the blood of carp. Cells were exposed to different MC-LR concentrations ranging from 0.01 to 1 µg ml(-1) for 2, 6 and 24 h. In addition, the effect of the toxin on the phagocytic activity of leukocytes and on actin and tubulin re-organization in phagocytic cells was studied. We observed that MC-LR induces apoptosis in lymphocytes 2 h after incubation, whereas high toxin concentrations induced necrosis in lymphocytes in a time- and concentration-dependent manner. Incubation of the cells for 2 h with 0.1 and 1 µg ml(-1) MC-LR inhibited phagocytosis without affecting apoptosis or glutathione (GSH) levels. Moreover, at this time point and with these concentrations, the toxin also induced a significant re-organization of the actin cytoskeleton in phagocytes, which subsequently collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. These results suggest that both phagocytes and lymphocytes are targets for MC-LR and the disturbances of phagocytosis may impair the balance of the immune system.

Pro- and anti-inflammatory cytokine expression in carp blood and head kidney leukocytes exposed to cyanotoxin stress--an in vitro study.

Cyanotoxins are toxic, secondary metabolites produced by different species of cyanobacteria that are present all over the world in aquatic environments. No data are available about the molecular mechanisms underlying the stress associated with exposure of fish immune cells to low concentrations of cyanotoxins. The purpose of this study was to determine whether the expression of cytokines that underlie immune regulation are changed after incubation of fish leukocytes with pure cyanotoxins: microcystin- LR (MC-LR), anatoxin-a (Antx-a), or an extract containing Antx-a. The study investigated the relative gene expression of four important cytokines, IL-1ß, TNF-α, IL-10, and TGF-ß, in carp head kidney and blood leukocytes exposed to toxins at concentrations of 0.01 or 0.1 µg/ml for 4 h. The data showed that pure toxins could induce dysregulation of pro-/anti-inflammatory cytokine expression. Expression of cytokine IL-1 ß was highly upregulated following Antx-a exposure, whereas MC-LR induced merely moderate reactions. The expression of TNF-α mRNA was significantly suppressed in blood and head kidney cells incubated with toxins at the higher concentration. These results showed that pure toxins dysregulated the expression of pro-inflammatory cytokines IL-1ß and TNF-α more promptly than the anti-inflammatory cytokines TGF-ß and IL-10. In contrast, the studies demonstrated a clearly downward trend of pro-inflammatory cytokines and an upward trend of anti-inflammatory cytokines in leukocytes exposed to an extract containing defined concentrations of Antx-a. This study suggests that cyanotoxins present in aquatic environments may exert immunotoxic effects by altering the transcription of important mediators of the fish immune system.

Effects of microcystin-containing cyanobacterial extract on hematological and biochemical parameters of common carp (Cyprinus carpio L.).

The aim of the study was to assess the effects of a cyanobacterial extract containing microcystins (MCs) on selected hematological and biochemical parameters in common carp (Cyprinus carpio L.), as well as to determine the accumulation of toxins in fish tissues. The fish were immersed for 5 days in water containing toxins at a final concentration of 12 µg/L of microcystin LR equivalent. Microcystin LR residues were detected in fish liver, reaching 207, 238 and 260 ng/g f.w. of the tissues taken 24 h, 72 h and 5 days after the end of intoxication, respectively. The most substantial changes were found in fish plasma, including increases in creatine kinase, lactate dehydrogenase, ammonia, glucose, aspartate aminotransferase and alanine aminotransferase levels. A decline of about 50% in lysozyme activity was observed by the end of the experimental period. Moreover, a marked increase in ceruloplasmin activity was detected 24 h after the end of intoxication with a subsequent decrease in its activity after 72 h and 5 days. This study concludes that not only consumption of food containing toxins but also MCs dissolved in water may pose a threat to fish health. Additionally, detected changes in lysozyme and ceruloplasmin activity may have distinct effects in fish resistance against pathogens or oxidative stress, which should be taken into account in the future studies.

Effects of neurotoxin - anatoxin-a on common carp (Cyprinus carpio L.) innate immune cells in vitro.

OBJECTIVES: The aim of this study was to determine if cyanoneurotoxin - anatoxin-a (ANTX-a) alters the essential functions of innate immune cells such as free radicals generation in phagocytic cells and phagocytosis. DESIGN: In the experiments pure ANTX-a was used at concentrations of 0.01, 0.05, 0.1 and 1 µg/ml RPMI-1640 medium. Phagocytes were isolated from carp blood and pronephros. Relative changes in intracellular total free radical presence in fish phagocytes were monitored using a fluorescent probe, dichlorodihydrofluorescin DiOxyQ (DCFH-DiOxyQ) which detects hydrogen peroxide (H2O2), nitric oxide (NO), peroxyl radical and peroxynitrite anion. Phagocytic activity of fish leukocytes was analyzed with a Vybrant phagocytosis assay kit. RESULTS: The H2O2 level generated in response to ANTX-a at the highest used concentration was significantly suppressed in pronephros but not in blood phagocytes. Moreover, it was observed that generation of superoxide radicals and nitrite formation was significantly increased in blood and pronephros phagocytes after incubation with lower concentrations of the neurotoxin. The phagocytosis of fish leukocytes was significantly reduced at the two highest used toxin concentrations (0.1 and 1 µg/ml medium). CONCLUSION: This findings suggests that ANTX-a could change innate immunity and reduced adaptive immunity after stress induced by cyanobacterial blooms.

Study on apoptotic effects of neurotoxin anatoxin-a on fish immune cells.

OBJECTIVES: The aim of this study was to assess the possible in vitro apoptotic effects of anatoxin-a on the selected immune cells isolated from the blood of carp. DESIGN: In the experiments pure anatoxin-a was used at concentrations of 0.01, 0.05, 0.1 and 1 µg/ml RPMI-1640 medium. Apoptosis or necrosis of fish leukocytes (lymphocytes and phagocytes) induced by the toxin was determined by measurement of the activity of caspases-3/7 and the analysis of phosphatidylserine on the outer leaflet of apoptotic cell-membranes using Annexin-V-Fluorescein and Propidium Iodide. Moreover, fluorescent measurement of the release of lactate dehydrogenase from the cells with damaged membranes was done. RESULTS: The viability of the lymphocytes exposed only to the highest concentration of anatoxin-a (1 µg/ml) was significantly decreased. The exposure to the toxin at higher concentrations (0.1 and 1 µg/ml) resulted in a significant increase of caspases 3/7 activity in phagocytes and lymphocytes. Moreover, fluorescent analysis with the use of annexin-V-fluorescein and propidium iodide staining showed more cells at the apoptotic stage than necrotic cells. CONCLUSION: Our study showed that anatoxin-a is an inducer of apoptosis in fish immune cells.

Evaluation of genotoxic potential of neurotoxin anatoxin-a with the use of umuC test.

OBJECTIVE: The aim of this study was to evaluate genotoxicity of anatoxin-a, cyanotoxin of neurotoxic activity. Additionally, other frequently detected cyanotoxin of previously described genotoxic potential, microcystin-LR, was used at the same concentrations, as well as the mixture of both toxins, anatoxin-a and microcystin-LR. DESIGN: Genotoxicity of the toxins was determined with the use of the umuC assay, in which the induction and expression of the umuC - lacZ reporter gene was assessed. The test was conducted on Salmonella typhimurium TA 1535/pSK1002 strain, with and without metabolic transformation. The toxin concentrations were 0.25, 0.5, 1 and 2 µg/ml. The exposure time was 2 h. RESULTS: The highest inefficient concentration of anatoxin-a without metabolic transformation was 0.25 µg/ml, of microcystin-LR was 0.5 µg/ml and in case of the toxin mixture all used concentrations induced the umuC gene. When S9 fraction was added to the samples, no effects were detected. CONCLUSION: To our knowledge, this is the first report on genotoxic effects of anatoxin-a. Although the study is preliminary and needs further research, however, indicates the new potential activity of the toxin, as well as the possible increase of genotoxicity of other cyanotoxins, more stable in the environment, e.g. microcystin-LR.

Toxicity of cyanobacterial bloom in the eutrophic dam reservoir (Southeast Poland).

Cyanobacterial bloom was observed in a highly eutrophic dam reservoir, Zemborzycki, near Lublin (SE Poland) over a warm period in the year 2007. The water bloom consisted of several cyanobacterial taxa: Anabaena circinalis, Anabaena spiroides, Anabaena flos-aquae, Planktothrix agardhii, Aphanizomenon flos-aquae, Aphanizomenon gracile, and Microcystis flos-aquae. Anabaena spp., and Aphanizomenon spp., potential producers of neurotoxic anatoxin-a, quantitatively predominated in the studied bloom. High-performance liquid chromatography (HPLC) analysis of surface scum sampled during Anabaena circinalis domination revealed the presence of anatoxin-a at a high concentration (1,035.59 microg per liter of surface scum). At the same time, neither gas chromatography/mass spectrometry (GC/MS) nor microcystin enzyme-linked immunosorbent assay (ELISA) test showed the presence of other frequently found cyanotoxins, microcystins. Toxicity of cyanobacterial bloom was assessed by the crustacean acute toxicity test Daphtoxkit F pulex using Daphnia pulex, and by the chronic toxicity test Protoxkit F with a ciliate protozoan Tetrahymena thermophila. The crude extract of cyanobacterial scum showed high toxicity for Daphnia pulex, with 24-h median effective concentration (EC50) value of 90.3 microg/L of anatoxin-a, which corresponded to the cyanobacterial density in the scum of 1.01 g dry weight/L. For Tetrahymena thermophila, 24-h EC50 was lower, evaluated to be 60.48 microg/L of anatoxin-a, which corresponded to a cyanobacterial density of 0.68 g dry weight/L of the scum. On the basis of evaluated toxicity units, the cyanobacterial extract was classified at class IV toxicity, which means high toxic hazard.