Cyanobacterial Crude Extracts Research Articles

Temperature rise and its influence on the toxic effects caused by cyanotoxins in a neotropical catfish

Potentially toxic cyanobacterial blooms (cyanoHABs) have become a problem in public water supply reservoirs. Temperature rise caused by climate change can increase the frequency and intensity of blooms, which may influence the cyanotoxins concentration in the environment. This study aimed to evaluate the effect of the temperature on the responses of a Neotropical catfish exposed to a neurotoxin-rich cyanobacterial crude extract (Raphidiopsis raciborskii T3). Juveniles of Rhamdia quelen were exposed to four treatments, based on study data: control at 25 °C (C25), control at 30 °C (C30), crude extract equivalent to 105 cells.mL-l of R. raciborskii at 25 °C (CE25) and 30 °C (CE30). After 96 h of exposure, the fish were anesthetized and blood was taken. After euthanasia, the gill, posterior kidney, brain, muscle, liver and gonad were sampled for hematological, biochemical, genotoxic and histopathological biomarker analysis. Liver was sampled for proteomic analysis for identification of proteins related to energy production. Water samples were collected at the beginning and the end of the experiment for neurotoxins quantification. Different parameters in both males and females were altered at CE25, evidencing the effects of neurotoxins in freshwater fish. At CE30, a water warming scenario, more effects were observed in females than at 25 °C, such as activation of saxitoxin metabolism pathway and genotoxicity. More damage to macromolecules was observed in females at the higher temperature, demonstrating that the increase in temperature can aggravate the toxicity of neurotoxins produced by R. raciborskii T3.

Liver dysfunction and energy storage mobilization in traíra, Hoplias malabaricus (Teleostei, Erythrinidae) induced by subchronic exposure to toxic cyanobacterial crude extract.

Microcystins (MC) are hepatotoxic for organisms. Liver MC accumulation and structural change are intensely studied, but the functional hepatic enzymes and energy metabolism have received little attention. This study investigated the liver and hepatocyte structures and the activity of key hepatic functional enzymes with emphasis on energetic metabolism changes after subchronic fish exposure to cyanobacterial crude extract (CE) containing MC. The Neotropical erythrinid fish, Hoplias malabaricus, were exposed intraperitoneally to CE containing 100 μg MC-LR eq kg-1 for 30 days and, thereafter, the plasma, liver, and white muscle was sampled for analyses. Liver tissue lost cellular structure organization showing round hepatocytes, hyperemia, and biliary duct obstruction. At the ultrastructural level, the mitochondria and the endoplasmic reticulum exhibited disorganization. Direct and total bilirubin increased in plasma. In the liver, the activity of acid phosphatase (ACP) increased, and the aspartate aminotransferase (AST) decreased; AST increased in plasma. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were unchanged in the liver, muscle, and plasma. Glycogen stores and the energetic metabolites as glucose, lactate, and pyruvate decrease in the liver; pyruvate decreased in plasma and lactate decreased in muscle. Ammonia levels increased and protein concentration decreased in plasma. CE alters liver morphology by causing hepatocyte intracellular disorder, obstructive cholestasis, and dysfunction in the activity of key liver enzymes. The increasing energy demand implies glucose mobilization and metabolic adjustments maintaining protein preservation and lipid recruitment to supply the needs for detoxification allowing fish survival.

Cucurbituril Ameliorates Liver Damage Induced by Microcystis aeruginosa in a Mouse Model.

Microcystis aeruginosa is a cyanobacterium that produces a variety of cyclic heptapeptide toxins in freshwater. The protective effects of the macromolecular container cucurbit[7]uril (CB7) were evaluated using mouse models of cyanotoxin-induced liver damage. Biochemical analysis of liver function was performed to gauge the extent of liver damage after exposure to cyanobacterial crude extract [CCE; LD50 = 35 mg/kg body weight; intraperitoneal (i.p.)] in the absence or presence of CB7 (35 mg/kg body weight, i.p.). CCE injection resulted in liver enlargement, potentiated the activities of alanine aminotransferase (ALT) and glutathione S-transferase (GST), increased lipid peroxidation (LPO), and reduced protein phosphatase 1 (PP1) activity. CCE-induced liver enlargement, ALT and GST activities, and LPO were significantly reduced when CB7 was coadministered. Moreover, the CCE-induced decline of PP1 activity was also ameliorated in the presence of CB7. Treatment with CB7 alone did not affect liver function, which exhibited a dose tolerance of 100 mg/kg body wt. Overall, our results illustrated that the addition of CB7 significantly reduced CCE-induced hepatotoxicity (P < 0.05).

Ecotoxicological investigation of cyanobacterial crude extracts to Daphniamagna under subchronic test conditions

Cyanobacterial blooms often consist of mixtures of microcystin-producing and microcystin-free species, and both can cause unpredictable effects on aquatic organisms. In this work, the subchronic effects of the cyanobacterial crude extracts (CCEs) from microcystin-producing and microcystin-free cyanobacteria with different microcystin concentrations (1, 10, and 50 μgL−1)on Daphnia magna were investigated. The life-history trait responses of D. magna to CCEs were determined based on survival, reproduction, and somatic growth. In addition, the physiological response, represented by the feeding rate of D. magna on green algae (Scenedesmussp.), after exposure to both types of crude extracts was estimated. Our results showed that both microcystin-containing (MCCE) and microcystin-free (NCCE) crude extracts insignificantly reduced survival but strongly enhanced reproduction and somatic growth of organisms. However, degradation of eggs and neonates of the gravid females exposed to CCEs was observed. In addition, the feeding rate of D. magna exposed to MCCE increased significantly, whereas no change in the feeding rate was observed for NCCE-exposed D. magna. In general, new and interesting aspects of the toxicity of MCCE and NCCE were revealed by this study, which contributes to the understanding of the toxicity of Pseudanabaena sp. extract on D. magna under bioassays.

A Long-Term Study on the Effect of Cyanobacterial Crude Extracts from Lake Chapultepec (Mexico City) on Selected Zooplankton Species.

Many urban lakes in Mexico City such as Lake Chapultepec are infested with high densities of cyanobacteria, particularly Microcystis. We tested the effect of cyanotoxins from cyanobacterial crude extracts on the demographic variables of zooplankton. The rotifers Brachionus havanaensis and Brachionus calyciflorus, and the cladocerans Ceriodaphnia dubia and Moina macrocopa were used for the assays. Temperature effects on the response of B. calyciflorus and 2 clones of M. macrocopa were tested. We hypothesized that with an increase in cyanotoxin concentration and temperature there would be an increase in the adverse effect on the test species and that the clone of Moina previously exposed to cyanobacteria from Lake Chapultepec would be more resistant to the cyanotoxins. Demography experiments showed that B. havanaensis was more sensitive than C. dubia. The negative effect of the cyanobacterial crude extract on B. calyciflorus was greater at 30 °C than at 20 °C or 25 °C. The strain of M. macrocopa isolated from Lake Chapultepec was more resistant to the cyanotoxins than the strain that had not been previously exposed to the cyanobacteria. The present study indicated that cyanobacteria in Lake Chapultepec are highly toxic and, considering the recreational use of this lake, should be controlled. Environ Toxicol Chem 2020;39:2409-2419. © 2020 SETAC.

DETECTION OF POTENTIALLY TOXIGENIC MICROCYSTIS STRAINS FROM DAU TIENG RESERVOIR

Microcystis is a bloom-forming, common cyanobacterium in Dau Tieng reservoir used for public water supply. To assess the presence of potentially microcystin-producing Microcystis, molecular techniques were conducted and acute toxicity bioassays were performed with the microcrustacean Daphnia magna exposed to cyanobacterial crude extracts. Potentially toxigenic of isolated strains was characterized by amplifying mcyD genes and identification of Microcystis was confirmed by 16S rRNA amplification. Microcystins (MCs) concentration in bloom samples and cultured strains were quantified by High Performance Liquid Chromatography (HPLC). Results showed that there were 9/15 strains showed positive with the mcyD marker indicating that they are toxic strains. Three MCs variants including MC-RR, -LR and -YR were found in all extracts of toxic strains with the highest concentration of 1,218 μg/g dry weight (DW). The acute toxicity bioassays revealed that both toxic and non-toxic crude extracts elicited significant lethal effects on the tested animal with LC50 values ranged from 189-411 mg DW/L. The toxic effects of isolated strains were independent from the MCs concentration in some strains suggesting the presence of other metabolites contributed to the biological effects. In conclusion, microcystin-producing Microcystis from the Dau Tieng reservoir warn about possible toxic effects for aquatic biota and human health.

Effects of two toxic cyanobacterial crude extracts containing microcystin-LR and cylindrospermopsin on the growth and photosynthetic capacity of the microalga Parachlorella kessleri

Secondary metabolites produced by cyanobacteria like microcystins (MC) and cylindrospermopsin (CYN) have been frequently studied because of their toxicity in humans and other animals. However, the function(s) of these metabolites remain largely unknown. The objective of this work was to deepen the knowledge in this research area and characterize the allelopathic effects of the cyanotoxins MC and CYN on the green alga Parachlorella kessleri. Several assays were carried out under controlled laboratory conditions with cyanobacterial cell extracts from Microcystis aeruginosa and Chrysosporum ovalisporum, producers of MC-LR and CYN respectively, at concentrations of 55 and 150 μg/L. The effects of CYN at 150 μg/L was also studied in P. kessleri growing in Z8 medium with altered nutrient composition. The data obtained indicate that growth rate and photosynthetic pigments were not affected by the exposure to cyanobacterial toxic extracts compared to control. However, the growth rate and photosynthetic pigments content changed during the 14 days of P. kessleri cultivation, in both groups (control and treatment), which may reflect the culture conditions and P. kessleri growth curve. It was also possible to verify that nutrients' concentration of the microalga culture medium does not influence the growth rate of the green microalga or the biological activity of the cyanobacterial extracts. In conclusion, under these specific laboratory conditions, there were no toxic or allelopathic effects of M. aeruginosa and C. ovalisporum crude extracts, which demonstrates the high tolerance of this phytoplanktonic species for the cyanobacterial toxins MC and CYN and other metabolites.

Effect of crude extracts from cyanobacterial blooms in Lake Texcoco (Mexico) on the population growth of Brachionus calyciflorus (Rotifera)

Unlike temperate regions, tropical ecosystems are characterized by high temperatures (>18 °C) all year, promoting blooms of cyanobacteria which often produce secondary metabolites toxic to zooplankton. Nabor Carillo and the Recreational Lake are part of the saline, Lake Texcoco, in Central Mexico which is filled nowadays with treated waste water. Both water bodies are dominated by Planktothrix, Anabaenopsis, Spirulina and Microcystis. In this study we present the concentration of microcystins in these waterbodies over an annual cycle. We also evaluated the chronic effects of cyanobacterial crude extracts from both lakes on two clones of the rotifer Brachionus calyciflorus, one from Nabor Carrillo Lake and the other from a canal in the shallow, Lake Xochimilco. The experiments on population growth were performed, beginning with 10 individuals per container for each of the following treatments: control (no crude extract), concentrated crude extract, and diluted crude extract (50:50) with moderately hard water and Chlorella vulgaris in a concentration of 0.5 × 106 cells ml−1. The cyanotoxin levels were measured using an ELISA test and ranged between 0.20 and 2.4 μg L−1 in the lake water. The results showed that the Recreational Lake extracts were more toxic, killing the rotifers in less than five days. The r values ranged from −1.74 to 0.48 in the presence of the crude extracts and 0.16 and 0.24 in the controls. The results have been discussed with emphasis on the importance of conducting regular studies to test ecotoxicological impacts of cyanobacterial blooms in tropical waters.

Effects of crude extracts of a saxitoxin-producer strain of the cyanobacterium Cylindrospermopsis raciborskii on the swimming behavior of wild and laboratory reared guppy Poecilia vivipara

The cyanobacterium Cylindrospermopsis raciborskii is an invasive species in water supply reservoirs worldwide, which can produces cylindrospermopsins and saxitoxins. In the wild, guppy (Poecilia vivipara) can be exposed to cyanotoxins, but those born and reared in laboratory are free of this contact. The aim of this paper was to comparatively measure the locomotor activity of ‘wild’ and ‘lab’ P. vivipara before and after exposure to crude extracts of two different cultures of C. raciborskii (CYRF-01), a saxitoxin-procucer strain. The movement of each fish was recorded using an image monitoring system (Videomex V®) before and after 48 h exposure to cyanobacterial extracts. Each experiment was performed during 4 h, with 1 h acclimation and 3 h recording period of the parameters Distance performed (DP), Swimming time (SwT), Stereotypic time (StT), Resting time (RT) and Average speed (AS). The quantification of saxitoxin in the solutions was performed by the enzyme-linked immunosorbent assay (ELISA). The weight or the total length did not influence the locomotor activity of fish in any of the experiments. The saxitoxin value was similar for both cultures (Culture 1: 7.3 μg L−1 and Culture 2: 8.6 μg L−1). However, in experiments with Culture 1 an increased activity in most parameters was observed, while in Culture 2, a decreased activity was observed only in ‘lab’ fish. Wild fish was less affected, showing higher resistance to both cyanobacterial crude extracts. This study showed that different cultures of the same strain of C. raciborskii and with similar contents of saxitoxin are able to change the locomotor activity of P. vivipara, contributing to the validation of the use of behavioral parameters to the evaluation of sublethal effects of toxic cyanobacteria on fish.

Microcystis extracts and single cells have differential impacts on the demography of cladocerans: a case study on Moina cf. micrura isolated from the Mediterranean coastal shallow lake (L’Albufera, Spain)

Cyanobacteria often have a deleterious effect on zooplankton. We hypothesized that the presence of either M. aeruginosa cells or microcystin extracts from M. aeruginosa would have a significant impact on the population growth rate, survivorship, and fecundity of Moina cf. micrura isolated from L’Albufera, Valencia, Spain. The cladocerans were exposed to different concentrations of Microcystis extracts on a diet of Nannochloris oculata (Chlorococcales) as well as different proportions of Microcystis single cells and N. oculata. Cyanotoxins were extracted from a Microcystis bloom by its repeated freezing, thawing, and sonication. Total microcystin concentration was 138.2 µg l−1. M. aeruginosa single cells were obtained by sonication. We used five microcystin concentrations (from 4.3 to 69.1 µg l−1 and controls without microcystins) with non-toxic Nannochloris oculata at 0.5 × 106 cells ml−1 as diet or on different proportions of M. aeruginosa single cells and N. oculata. Microcystin reduced generation time and longevity of M. cf. micrura but increased daily production of offspring. Single-celled Microcystis (100%) diet for Moina cf. micrura decreased growth rates compared with treatments using cyanotoxin extracts. We suggest routine testing of cyanobacterial crude extracts using plankton to estimate harmful impacts of blooms especially for drinking water reservoirs.

Lettuce irrigated with contaminated water: Photosynthetic effects, antioxidative response and bioaccumulation of microcystin congeners

The use of microcystins (MCs) contaminated water to irrigate crop plants represents a human health risk due to their bioaccumulation potential. In addition, MCs cause oxidative stress and negatively influence photosynthetic activities in plants. The present study was aimed at investigating the effect of MCs on photosynthetic parameters and antioxidative response of lettuce. Furthermore, the bioaccumulation factor (BAF) of total MCs, MC-LR and MC-RR in the vegetable after irrigation with contaminated water was determined. Lettuce crops were irrigated for 15 days with water containing cyanobacterial crude extracts (Microcystis aeruginosa) with MC-LR (0.0, 0.5, 2.0, 5.0 and 10.0µgL−1), MC-RR (0.0, 0.15, 0.5, 1.5 and 3.0µgL−1) and total MCs (0.0, 0.65, 2.5, 6.5 and 13.0µgL−1). Increased net photosynthetic rate, stomatal conductance, leaf tissue transpiration and intercellular CO2 concentration were recorded in lettuce exposed to different MCs concentrations. Antioxidant response showed that glutathione S-transferase activity was down-regulated in the presence of MCs. On the other hand, superoxide dismutase, catalase and peroxidase activities were upregulated with increasing MCs concentrations. The bioaccumulation factor (BAF) of total MCs and MC-LR was highest at 6.50 and 5.00µgL−1, respectively, while for MC-RR, the highest BAF was recorded at 1.50µgL−1 concentration. The amount of total MCs, MC-LR and MC-RR bioacumulated in lettuce was highest at the highest exposure concentrations. However, at the lowest exposure concentration, there were no detectable levels of MC-LR, MC-RR and total MCs in lettuce. Thus, the bioaccumulation of MCs in lettuce varies according to the exposure concentration. In addition, the extent of physiological response of lettuce to the toxins relies on exposure concentrations.

Microcystin accumulation and biochemical responses in the edible clam Corbiculaleana P. exposed to cyanobacterial crude extract

We investigated the accumulation and effects of cyanobacterial crude extract (CCE) containing microcystins (MCs) on the edible clam Corbiculaleana P. Toxic effects were evaluated through the activity of antioxidant and detoxification enzymes: catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferases (GSTs) from gills, foot, mantle and remaining soft tissues. Clams were exposed to CCE containing 400μg MC-LReq/L for 10days and were then kept in toxin-free water for 5days. Clam accumulated MCs (up to 3.41±0.63μg/g dry weight (DW) of unbound MC and 0.31±0.013μg/g DW of covalently bound MC). Detoxification and antioxidant enzymes in different organs responded differently to CCE during the experiment. The activity of SOD, CAT, and GST in the gills and mantle increased in MC-treated clams. In contrast, CAT and GST activity was significantly inhibited in the foot and mostly only slightly changed in the remaining tissues. The responses of biotransformation, antioxidant enzyme activity to CCE and the fast elimination of MCs during depuration help to explain how the clam can survive for long periods (over a week) during the decay of toxic cyanobacterial blooms in nature.

Cytotoxicity evaluation of large cyanobacterial strain set using selected human and murine in vitro cell models

The production of cytotoxic molecules interfering with mammalian cells is extensively reported in cyanobacteria. These compounds may have a use in pharmacological applications; however, their potential toxicity needs to be considered. We performed cytotoxicity tests of crude cyanobacterial extracts in six cell models in order to address the frequency of cyanobacterial cytotoxicity to human cells and the level of specificity to a particular cell line. A set of more than 100 cyanobacterial crude extracts isolated from soil habitats (mainly genera Nostoc and Tolypothrix) was tested by MTT test for in vitro toxicity on the hepatic and non-hepatic human cell lines HepG2 and HeLa, and three cell systems of rodent origin: Yac-1, Sp-2 and Balb/c 3T3 fibroblasts. Furthermore, a subset of the extracts was assessed for cytotoxicity against primary cultures of human hepatocytes as a model for evaluating potential hepatotoxicity. Roughly one third of cyanobacterial extracts caused cytotoxic effects (i.e. viability<75%) on human cell lines. Despite the sensitivity differences, high correlation coefficients among the inhibition values were obtained for particular cell systems. This suggests a prevailing general cytotoxic effect of extracts and their constituents. The non-transformed immortalized fibroblasts (Balb/c 3T3) and hepatic cancer line HepG2 exhibited good correlations with primary cultures of human hepatocytes. The presence of cytotoxic fractions in strongly cytotoxic extracts was confirmed by an activity-guided HPLC fractionation, and it was demonstrated that cyanobacterial cytotoxicity is caused by a mixture of components with similar hydrophobic/hydrophilic properties. The data presented here could be used in further research into in vitro testing based on human models for the toxicological monitoring of complex cyanobacterial samples.

Effects of Cylindrospermopsin Producing Cyanobacterium and Its Crude Extracts on a Benthic Green Alga-Competition or Allelopathy?

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by filamentous cyanobacteria which could work as an allelopathic substance, although its ecological role in cyanobacterial-algal assemblages is mostly unclear. The competition between the CYN-producing cyanobacterium Chrysosporum (Aphanizomenon) ovalisporum, and the benthic green alga Chlorococcum sp. was investigated in mixed cultures, and the effects of CYN-containing cyanobacterial crude extract on Chlorococcum sp. were tested by treatments with crude extracts containing total cell debris, and with cell debris free crude extracts, modelling the collapse of a cyanobacterial water bloom. The growth inhibition of Chlorococcum sp. increased with the increasing ratio of the cyanobacterium in mixed cultures (inhibition ranged from 26% to 87% compared to control). Interestingly, inhibition of the cyanobacterium growth also occurred in mixed cultures, and it was more pronounced than it was expected. The inhibitory effects of cyanobacterial crude extracts on Chlorococcum cultures were concentration-dependent. The presence of C. ovalisporum in mixed cultures did not cause significant differences in nutrient content compared to Chlorococcum control culture, so the growth inhibition of the green alga could be linked to the presence of CYN and/or other bioactive compounds.

Effects of the naturally-occurring contaminant microcystins on the Azolla filiculoides–Anabaena azollae symbiosis

Harmful algal blooms (HABs) contaminate aquatic ecosystems and are responsible for animal poisoning worldwide. We conducted a toxicity test with the aquatic fern and the biofertilizer, Azolla filiculoides. The sporophytes were exposed to three concentrations (0.01, 0.1 and 1μgmL−1) of a microcystin (MC) cyanobacterial crude extract and purified MC-LR. The growth of A. filiculoides decreased only at 1μgmL−1 crude extract concentration while with MC-LR it decreased at all the tested concentrations, indicating that the presence of other compounds in the crude extract altered toxicity and stimulated the fern growth at lower concentrations (0.01 and 0.1μgmL−1). Both phycoerythrocyanin and allophycocyanin levels decreased in all the concentrations of crude extract and MC-LR. The phycocyanin had a marked increase at 0.1μgmL−1 crude extract concentration and a marked decrease at 1μgmL−1 MC-LR concentration. These changes in the phycobiliprotein content indicate a shift in the antenna pigments of the cyanobionts of A. filiculoides. The changes in two oxidative stress enzymes, glutathione reductase for the crude extract assay and glutathione peroxidase for MC-LR assay, points towards the induction of stress defense responses. The low bioconcentration factor in both crude extract and MC-LR treatments can suggest the low uptake of microcystins, and indicates that the aquatic fern can be used as a biofertilizer and as animal feed but is not suitable for MC phytoremediation.

Effect of crude extracts of Dolichospermum planctonicum on the demography of Plationus patulus (Rotifera) and Ceriodaphnia cornuta (Cladocera).

In this study we evaluated the toxicological effects of crude extracts of Dolichospermum planctonicum on Plationus patulus (Rotifera) and Ceriodaphnia cornuta (Cladocera). The active compound from the laboratory-cultured cyanobacteria was extracted by lyophilizing it at -70°C for 48h, after which the sample was thawed. The cycle was repeated five times, and between each cycle the cells were sonicated to lyse them. Using the extract of Anabaena, experiments of population growth and life table demography of the two zooplankton species were compared in media with and without (controls) the cyanotoxins. In both experiments two toxic concentrations diluted in US EPA medium were used, one in a 5:15 proportion, and the other in a 2.5:17.5. We found that the rotifers were more resistant to the toxicants than the cladocerans. Their population growth rates ranged from 0.11d(-1) in controls to 0.08d(-1) in the presence of the crude extracts for P. patulus and from 0.18 in controls to -0.27 in the presence of the crude extracts for C. cornuta. The crude extract also had a significant negative impact on the demographic parameters, adversely affecting reproduction more than survival. The data have been discussed with emphasis on the importance of further research using cyanobacterial crude extracts.

Evaluation of the Impact of Natural Microcystin-LR Extract on Brine Shrimp as an Invertebrate Bioassay Model

Frequent occurrences of toxic cyanobacteria dominantly Microcystis aeruginosa bloom arebecoming a nuisance problem in Sri Lankan reservoirs in conformity with its globalenvironmental and health issues. Most of cyanobacteria produce heat stable cyanotoxinswhich have potent health impacts. Many researchers have focused on pure cyanobacterialtoxins and little was known about the toxic effect of natural cyanobacteria. The present studywas carried out to investigate the effect of natural cyanobacterial toxins on Artemia salina(Brine shrimp) as a bioassay. The toxin, Microcystin LR was extracted from bloom samplescollected from Beira and Borelesgamuwa Lakes. Both water bodies were dominated bycyanobacterium M. aeruginosa, which may contain toxic and non toxic strains. Density of M.aeruginosa at the sampling occasion in Beira Lake was 9.64x105cells/l and inBoralesgamuwa Lake was 5.31x105 cells/l respectively. One liter of lake water was filteredthrough GF/C glass microfiber filters (47 mm) and the filter disks with entrappedcyanobacterial cells were ultrasonicated with 20 ml of 100% methanol for 5 minutes and thenthe methanolic extract was filtered and HPLC-DAD analysis was done to quantify theMicrocystin (MC)-LR. MC-LR in Beira Lake was 17.13±0.02 μg/ml. In BorelesgamuwaLake MC-LR was not detected at the sampling occasion. Methanol in the extract wasevaporated and subsequently a known weight of residue was re-suspended in 2 ml of filteredsea water to make stock solutions of crude extracts to use for the bioassay. Artemia eggs werepurchased and allowed to hatch with continuous aeration in filtered sea water undercontinuous white fluorescent light for 18 h. After incubation newly hatched naupli of Artemiawere collected. For the bioassay about 30 Artemia naupli were inoculated into each well in96-well microplate. To confirm the initial number of naupli in each well, counting wasperformed under the microscope. MC-LR standard and extracts from Beira andBoralesgamuwa Lakes were used to prepare toxin (MC-LR) at final concentration series of0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0 and 50.0 μg/ml. Number of dead individuals were counted ineach well after 6, 12, 18, 24 and 48 hours incubation. EC50 (50% effective concentration)values were assessed using non-linear regression by Graph Pad Prism software. EC50 value ofMC-LR standard was 4.78 μg/ml after 6 h incubation. However, EC50 values ofcyanobacterial crude extracts from Beira and Boralesgamuwa lakes at the same incubationperiod was 4.98 and 9.83 μg dry weight/ml respectively. The results showed that the standardMC-LR and cyanobacterial crude extracts from Beira Lake had almost similar EC50 valuewhile Boralesgamuwa Lakes was significantly deviated and having higher (p&lt;0.05) EC50value. The higher toxicity of Beira Lake crude extract was due to presence of MC-LR in theextract. As confirmed by the HPLC, MC-LR was not detected in the extract prepared fromBoralesgamuwa Lake. Though the extracts showed toxicity effect on Artemia reveal that thetoxicity may due to other cyanotoxin variants present in the extracts. Further, it was detectedthat decreasing tendency of EC50 values along with the increasing incubation periods for allextracts and standard.

Physiological and biochemical defense reactions of Vicia faba L.–Rhizobium symbiosis face to chronic exposure to cyanobacterial bloom extract containing microcystins

The presence of cyanotoxins, mainly microcystins (MCs), in surface freshwater represents a serious health risk to aquatic organisms living in the water body, as well as terrestrial animals and plants that are in contact with contaminated water. Consequently, the use of MCs contaminated water for irrigation represents a hazard for cultivated plants and could induce severe economical losses due to crops' yield reduction. The experimental approach undertaken in this work was exposing Vicia faba seedlings (inoculated with a Rhizobium strain resistant to MCs), to water supplemented with cyanobacterial crude extract containing total microcystins at a concentration of 50 and 100 μg/L (environmental relevant concentrations of MCs dissolved in the raw irrigation water from Lalla Takerkoust Lake-Marrakesh region). After chronic MCs exposure (2 months), biological and physiological parameters (plant growth, nitrogen uptake, mineral assimilation, and oxidative defense mechanisms) were evaluated. The results obtained showed evidence that chronic exposure to cyanobacterial bloom extract containing MCs strongly affected the physiological and biological plants activities; reduction of dry matter, photosynthetic activity, nodule number, and nitrogen assimilation. At the same time, an increase of oxidative stress was observed, as deduced from a significant increase of the activities of peroxidase, catalase, polyphenoloxidase, and phenylalanine ammonia lyase in leaves, roots, and nodules of faba bean plants exposed to cyanotoxins, especially at 100 μg/L of MCs. This experimentation constitutes a simulation of the situation related to cyanotoxins chronic exposure of seedlings-plants via the contaminated irrigation water. For this reason, once should take into consideration the possibility of contamination of agricultural crops and the quality of irrigation water should be by the way monitored for cyanotoxins biohazard.

Absence of negative allelopathic effects of cylindrospermopsin and microcystin-LR on selected marine and freshwater phytoplankton species

Cyanobacterial toxins have been regarded by some researchers as allelopathic substances that could modulate the growth of competitors. Nevertheless, often the concentrations of toxins used are too high to be considered ecologically relevant. In this work we tested the hypothesis that microcystin-LR (MC-LR) and cylindrospermopsin (CYN) at ecologically relevant concentrations have no allelopathic effects on some species of phytoplankton. Extracts containing the toxins as well as pure MC-LR and CYN toxins were used to assess their effects on the growth rates of Nannochloropsis sp., Chlamydomonas reinhardtii, and Chlorella vulgaris. Cyanobacterial crude extracts induced more pronounced effects on growth rates than pure toxins. Microcystis aeruginosa and Aphanizomenon ovalisporum crude extracts containing MC-LR and CYN at 0.025–2.5 mg l−1 stimulated growth rates of microalgae, whereas A. ovalisporum crude extracts containing 2.5 mg l−1 of CYN strongly inhibited growth rates of microalgae after 4 and 7 days of exposure. MC-LR and CYN at environmentally occurring concentrations were unable to affect negatively the growth of microalgae, and therefore these molecules may play roles other than allelopathy in natural ecosystems.

Adaptation of freshwater mussels to cyanobacterial toxins: Response of the biotransformation and antioxidant enzymes

Freshwater mussels such as the invasive Dreissena polymorpha and the indigenous Unio tumidus nourish by high filtration rates and may accumulate cyanobacteria and their toxins during cyanobacterial blooms. Physiological adaptations to cyanotoxins enable organisms to endure cyanobacterial blooms but may differ between species. Biotransformation and excretion capacities for cyanobacteria and anthropogenic pollutants have been demonstrated for Dreissena polymorpha but less for unionid species. This study compares the activities of biotransformation (glutathione S-transferase, GST) and antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) in Dreissena polymorpha to Unio tumidus in response to cyanotoxin exposure (10μgL−1 and 50μgL−1 microcystin-LR, respectively, total microcystin from a cyanobacterial crude extract) for 24h and 7d exposure duration. Enzyme activities in Dreissena polymorpha were measured in the whole mussel tissue, digestive gland and in gills and in Unio tumidus in the digestive gland, gills, mantle, foot as well as in the remaining tissue. The sGST was elevated for the entire exposure period in the whole mussel tissue of Dreissena polymorpha but despite higher basal activities in digestive gland and gills of Unio tumidus, it was rather inhibited or unaltered in most of their tissues. Elevated SOD activity indicated oxidative stress response in Dreissena polymorpha, but not in Unio tumidus. The CAT activity was barely affected in both species, rather inhibited in Unio tumidus, despite again higher basal activities in digestive gland and remaining tissue. Compared to the indigenous Unio tumidus, the investigated biotransformation and oxidative stress combating enzymes respond stronger in the invasive Dreissena polymorpha.