Occurrence of the cyanobacterial toxin cylindrospermopsin in northeast Germany

The frequent occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) in the (sub)tropics has been largely associated with cyanobacteria of the order Nostocales of tropical origin, in particular Cylindrospermopsis raciborskii. C. raciborskii is currently observed to spread northwards into temperate climatic zones. In addition, further cyanobacteria of the order Nostocales typically inhabiting water bodies in temperate regions are being identified as CYN-producers. Therefore, data on the distribution of CYN in temperate regions are necessary for a first assessment of potential risks due to CYN in water used for drinking and recreation. A total of 127 lakes situated in the north-eastern part of Germany were investigated in 2004 for the presence of the toxin CYN and the phytoplankton composition. The toxin could be detected in half of the lakes (n = 63) and in half of 165 samples (n = 88). Concentrations reached up to 73.2 microg CYN/g DW. CYN thus proved more widely distributed than previously demonstrated. The analyses of phytoplankton data suggest Aphanizomenon sp. and Anabaena sp. as important CYN producers in Germany, and confirm recent findings of Aphanizomenon flos-aquae as CYN-producing species frequently inhabiting water bodies in temperate climatic regions. The data shown here suggest that CYN may be an important cyanobacterial toxin in German water bodies and that further data are needed to assess this.     

Multiple-organ toxicity resulting from cylindrospermopsin exposure in tadpoles of the cane toad (Bufo marinus)

Histological examinations were made of cane toad (Bufo marinus) tadpoles after exposure to freeze-thawed Cylindrospermopsis raciborskii whole cell extracts and live C. raciborskii cultures containing sublethal concentrations of the blue-green algal toxin, cylindrospermopsin (CYN). Toxin exposure resulted in tissue injuries to multiple organs, with particular severity noted in the liver, intestine, nephric ducts and gill epithelia. The extent of cellular damage was similar across trials exposing tadpoles to aqueous and cell-bound toxins, despite unequal toxin concentrations being present in each. It was concluded that the presence of cell-bound toxin, which may be directly ingested via grazing, plays a crucial role in the exertion of histological effects in B. marinus. This work provides baseline information regarding the ecotoxicity of CYN toward amphibians. The range of cellular effects noted in CYN-exposed tadpoles suggests that toxic C. raciborskii blooms could represent considerable health risks to amphibian populations and indicate potentially far-reaching ecological impacts of toxic C. raciborskii blooms.     

Alternative bioassay for the detection of saxitoxin using the speckled cockroach (Nauphoeta cinerea)

Paralytic shellfish poisoning (PSP) toxins produced by cyanobacteria pose a risk to public health as they occur in drinking water reservoirs and recreational lakes and accumulate in the food chain. One of these PSP toxins, saxitoxin (STX) is one of the most toxic nonprotein substances known. Accordingly, there is a requirement to monitor for these toxins. The standard bioassay used to detect these toxins is the mouse bioassay; however, its use is constrained by animal ethics guidelines and practical considerations. Reported here is the use of the globally distributed speckled cockroach Nauphoeta cinerea as a bioassay test organism for the selective detection of PSP toxicity of Anabaena circinalis aqueous extract and STX. N. cinerea was shown to be tolerant to pure cylindrospermopsin (CYN) and microcystin-LR (MC-LR) at doses 10-fold greater than mouse LD?? values while being sensitive to STX. Similarly, N. cinerea was shown to be tolerant of toxin-containing aqueous extracts of Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Nodularia spumigena while being sensitive to A. circinalis. Peak sensitivity to STX was 60 min postinjection with a KD?? of 31.2 ng/g body weight. While this was approximately 3-fold less sensitive than the mouse bioassay, the insect test organism was around 34-fold smaller in mass than a mouse (20 g); thus one-tenth the amount of toxin in absolute quantity was required to reach an ED?? level. The N. cinerea bioassay presents a selective test for PSP toxicity that is rapid, economical, efficient, and simple to perform.     

The Palm Island mystery disease 20 years on: a review of research on the cyanotoxin cylindrospermopsin

Poisoning of humans resulting from consumption of water affected by the toxic cyanobacterium Cylindrospermopsis raciborskii was first reported almost 20 years ago from Palm Island, northern Queensland, Australia. Since that time a great deal has been learned about this organism and cylindrospermopsin (CYN), the toxin it produces. This article reviews the information now available to us. It summarizes aspects of the chemistry of the toxin-now known to be produced by some cyanobacterial species other than C. raciborskii-and its biosynthesis and chemical synthesis in vitro, as well as its detection and measurement by chemical and biological assay. Some of the factors affecting toxin production by cultured isolates of C. raciborskii are reviewed and the conditions that cause its release from the cells described. The occurrence of CYN in water bodies and the management strategies used to minimize the harmful effects of the toxin are outlined. These include a range of water-treatment practices now in place to remove CYN-producing organisms and/or to neutralize the toxin together with some management procedures that have been tried, with varying degrees of success, to prevent buildup of blooms of the offending organisms. Some of the public-health considerations arising from exposure to water supplies affected by CYN are summarized along with the risk factors and guidance values as they are currently applied. Among the more recent developments described are those that come from the application of molecular techniques for characterizing toxic and nontoxic strains and for exploring the genetic aspects of CYN production.     

Occurrence and elimination of cyanobacterial toxins in two Australian drinking water treatment plants.

In Australian freshwaters, Anabaena circinalis, Microcystis spp. and Cylindrospermopsis raciborskii are the dominant toxic cyanobacteria. Many of these surface waters are used as drinking water resources. Therefore, the National Health and Medical Research Council of Australia set a guideline for MC-LR toxicity equivalents of 1.3 microg/l drinking water. However, due to lack of adequate data, no guideline values for paralytic shellfish poisons (PSPs) (e.g. saxitoxins) or cylindrospermopsin (CYN) have been set. In this spot check, the concentration of microcystins (MCs), PSPs and CYN were determined by ADDA-ELISA, cPPA, HPLC-DAD and/or HPLC-MS/MS, respectively, in two water treatment plants in Queensland/Australia and compared to phytoplankton data collected by Queensland Health, Brisbane. Depending on the predominant cyanobacterial species in a bloom, concentrations of up to 8.0, 17.0 and 1.3 microg/l were found for MCs, PSPs and CYN, respectively. However, only traces (<1.0 microg/l) of these toxins were detected in final water (final product of the drinking water treatment plant) and tap water (household sample). Despite the low concentrations of toxins detected in drinking water, a further reduction of cyanobacterial toxins is recommended to guarantee public safety.     

Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes.

The cyanobacterial toxin cylindrospermopsin (CYN) is widely distributed in German lakes, but volumetric data for risk assessment are lacking and it is unclear which cyanobacterial species produce CYN in Europe. We therefore analyzed CYN concentration and cyanobacterial composition of 21 German lakes in 2005. CYN was detected in 19 lakes (102 of 115 samples). In total, 45 samples contained particulate CYN only, and 57 contained both dissolved and particulate CYN. The concentrations were 0.002-0.484 microg L(-1) for particulate CYN and 0.08-11.75 microg L(-1) for dissolved CYN with a maximum of 12.1 microg L(-1) total CYN. A drinking water guideline value of 1 microg L(-1) proposed by Humpage and Falconer [2003. Oral toxicity of the cyanobacterial toxin CYN in male Swiss albino mice: determination of no observed adverse effect level for deriving a drinking water guideline value. Environ. Toxicol. 18, 94-103] was exceeded in 18 samples from eight lakes due to high concentrations of dissolved CYN. CYN occurrence in the German lakes could not be ascribed to the three known CYN-producing species Cylindrospermopsis raciborskii, Anabaena bergii and Aphanizomenon flos-aquae, which were detected in some lakes in low abundances. The highest correlation coefficients were observed between particulate CYN and the native Aphanizomenon gracile. It occurred in 98 CYN-positive samples, was the most abundant Nostocales and was the only Nostocales in five samples. This indicates that A. gracile is a potential CYN producer in German lakes.     

Influence of intracellular toxin concentrations on cylindrospermopsin bioaccumulation in a freshwater gastropod (Melanoides tuberculata).

Scant information is available regarding the bioaccumulation of cylindrospermopsin (CYN) in aquatic organisms, particularly in invertebrates. This study examined toxin bioconcentration and bioaccumulation in the aquatic snail, Melanoides tuberculata, following exposure to freeze-thawed whole cell extracts and a live Cylindrospermopsis raciborskii culture containing CYN. Both bioconcentration and bioaccumulation were evident, but exposure to toxin in the freeze-thawed solutions resulted in minor tissue contamination compared with that resulting from live C. raciborskii exposure. Thus, whilst CYN uptake resulted from both extracellular and intracellular exposures, the availability of intracellular toxin was critical in affecting tissue CYN values. M. tuberculata did not bioconcentrate CYN into the shell. Bioaccumulation of the analog deoxy-CYN was also recorded. Knowledge of intracellular toxin concentrations may be critical in evaluating the bioaccumulation, ecological and human health risks associated with contaminated systems.     

Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria).

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250mg dry weight cells/kg body weight within 24h and 125mg/kg at 72h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262nm. HPLC-MS/MS confirmed the presence of CYN (M+H 416) and deoxy-CYN (M+H 400). The CYN content in strain CY-Thai was estimated at 1.02mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii.     

A comparative study of Florida strains of Cylindrospermopsis and Aphanizomenon for cylindrospermopsin production.

The toxin cylindrospermopsin (CYN) is produced by a variety of cyanobacterial genera. One of these, Cylindrospermopsis raciborskii, is generally assumed to be the source of CYN in lakes and rivers in Florida, USA. However, in this study, none of the eight Florida isolates of this species tested contained the genetic determinants involved in toxin production nor did they produce CYN. We show for the first time that Aphanizomenon ovalisporum isolated from a pond in this state has the genes putatively associated with CYN production. Analysis by liquid chromatography with mass spectrometric detection (LC/MS) revealed that it produced CYN in the range of 7.39-9.33 microg mg(-1) freeze-dried cells. 16S rDNA sequences of this strain showed 99.6% and 99.9% identity to published A. ovalisporum and Anabaena bergii 16S sequences, respectively. These results help to explain the general lack of a defined relationship between the abundance of C. raciborskii in freshwater ecosystems of Florida and observed concentrations of CYN. The latter observation raises the potential that previous reports of CYN may be coincidental with unrecorded presence of another CYN-producing species.     

Toxicological comparison of diverse Cylindrospermopsis raciborskii strains: evidence of liver damage caused by a French C raciborskii strain

The freshwater cyanobacterium Cylindrospermopsis raciborskii is known to produce toxic effects in several countries. Acute and chronic exposures to C. raciborskii in Australia have been linked to liver damage (hepatotoxicity) with concomitant effects on the kidneys, adrenal glands, small intestine, lungs, thymus, and heart. The alkaloid cylindrospermopsin, which produces these toxic effects, is thought to be a potent inhibitor of protein synthesis. C. raciborskii strains producing cylindrospermopsin or analogue alkaloids have also been reported in Florida, USA, and Thailand. Brazilian isolates of C. raciborskii are also toxic but act by a different mechanism, causing acute death in mice with neurotoxic symptoms similar to those induced by the saxitoxins. In this article we compare the toxicity in the mouse of a C. raciborskii French strain with C. raciborskii strains from various other sources (Australia, Brazil, Mexico, and Hungary). We tested the toxicity of cell extracts by a mouse bioassay. Acute, fatal neurotoxicity was produced by the Brazilian strain, which was confirmed by liquid chromatography with fluorescence detection of the cell extracts, which revealed the presence of saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin, along with two unidentified compounds. Acute hepatotoxicity with severe liver, kidney, and thymus damage was observed with the Australian cylindrospermopsin-producing strain. The Mexican and Hungarian strains were not found to be toxic to mice in our experimental conditions. No animals died after exposure to the extracts of the French C. raciborskii strain. Histological examination of the liver revealed moderate, multifocal necrosis characterized by small areas of hepatocellular necrosis, combined with disorganization of the parenchyma and congestion of the inner sinusoid. These symptoms and lesions resembled those induced by cylindrospermopsin, but the chemical analysis performed by liquid chromatography coupled with either a diode array detector or a mass spectrometer demonstrated that this toxin was not present in our culture extract.     

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.     

Elucidating the factors influencing the biodegradation of cylindrospermopsin in drinking water sources

The cyanotoxin cylindrospermopsin (CYN) is produced by several species of cyanobacteria and can be persistent in drinking waters supplies, which is of major concern to water authorities because of its potential to severely compromise human health. Consequently, there is a need to fully understand the persistence of CYN in water supplies, in particular, to determine whether this toxin is readily degraded by endemic aquatic organisms. This study provides insights into the environmental factors that can influence the biodegradation of this toxin in Australian drinking water supplies. Biodegradation of CYN was only evident in water supplies that had a history of toxic Cylindrospermopsis raciborskii blooms. In addition, lag periods were evident prior to the onset of biodegradation; however, repeated exposure of the endemic organisms to CYN resulted in substantial decreases in the lag periods. Furthermore, the concentration of CYN was shown to influence biodegradation with a near linear relationship (R(2) of 0.9549) existing between the biodegradation rate and the initial CYN concentration. Temperature was also shown to affect the biodegradation of CYN, which is important since CYN is now being detected in more temperate climates. The presence of copper-based algicides inhibited CYN degradation, which has significant implications since copper-based algicides are commonly used to control cyanobacterial growth in water bodies. The results from this study indicate that the biodegradation of CYN in natural water bodies is a complex process that can be influenced by many environmental factors, some of which include CYN concentration, temperature, and the presence of copper-based algicides.     

Accumulation and depuration of the cyanobacterial toxin cylindrospermopsin in the freshwater mussel Anodonta cygnea.

Cylindrospermopsin (CYN) is a toxic alkaloid produced by several genera of freshwater cyanobacteria. This compound has been implicated in outbreaks of human sickness and the death of domestic and wild animals. Given that several of the cyanobacterial genera known to produce CYN are common components of the phytoplankton of freshwaters including aquaculture facilities, we studied the accumulation of CYN in the freshwater mussel (swan mussel) Anodonta cygnea. Anodonta were exposed to CYN-producing cultures of the cyanobacterium Cylindrospermopsis raciborskii for 16 days and were found to accumulate the toxin to concentrations up to 2.52 microg g tissue dry weight(-1). There was considerable variation in the concentrations of CYN detected in different parts of the body. At the end of a 2-week accumulation period the distribution of CYN in the body of Anodonta was as follows: haemolymph (68.1%), viscera (23.3%), foot and gonad (7.7%) and mantle (0.9%). No CYN was detected in the gills or adductor muscle of any animals. Following a 2-week depuration period, approximately 50% of the toxin remained in the tissues. Based on the recently derived guideline value for CYN in human drinking water (1 microg l(-1)) and the concentrations of this compound in animal tissues reported here, there is a clear need for the increased monitoring of this compound in organisms grown for human and animal consumption.     

The effect of temperature on growth and production of paralytic shellfish poisoning toxins by the cyanobacterium Cylindrospermopsis raciborskii C10.

Cylindrospermopsis raciborskii is a cyanobacterium which produces either cylindrospermopsine or paralytic shellfish poisoning (PSP) toxins. We studied the effect of temperature on growth and production of PSP toxins by C. raciborskii C10, isolated from a freshwater reservoir in Brazil. We analyzed the extracellular and intracellular content of PSP toxins at two different temperatures: 19 and 25 degrees C. C. raciborskii C10 produces STX, GTX2, and GTX3 at both temperatures. dcSTX was also detected at 25 degrees C in the intracellular extracts obtained at the end of the stationary phase. The growth achieved at 25 degrees C and estimated by optical density at 700 nm was three times greater than at 19 degrees C. However, no significant differences were observed in the content of PSP toxins in either the cells or the extracellular media. The kinetics of accumulation of PSP toxins within the cells rather than in the media suggests an active PSP toxins-export process that is not related to cell lysis. The extracellular accumulation of PSP toxins at 19 degrees C suggested a biotransformation of STX to the epimers GTX2 and GTX3. The stability of the PSP toxins produced by C. raciborskii C10 was high enough for them to remain active in the media after 30 days (at 25 degrees C) or after 50 days (at 19 degrees C).     

The first evidence of paralytic shellfish toxins in the fresh water cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil.

The blooms of toxic cyanobacteria (blue-green algae) are causing problems in many countries. During a screening of toxic freshwater cyanobacteria in Brazil, three strains isolated from the State of Sao Paulo were found toxic by the mouse bioassay. They all were identified as Cylindrospermopsis raciborskii by a close morphological examination. Extracts of cultured cells caused acute death to mice when injected intraperitoneally after developing neurotoxic symptoms which resembled to those caused by paralytic shellfish toxins. The analysis of the sample by HPLC-FLD postcolumn derivatization method for paralytic shellfish toxins resulted in the detection of several saxitoxin analogs. To avoid being misled by false peaks, the sample was reanalyzed after purification and also under the different postcolumn derivatizing conditions. Finally, the newly developed LC-MS method for paralytic shellfish toxins was applied to unambiguously identify the toxins. One isolate produced neosaxitoxin predominantly with saxitoxin as a minor component. The other two showed identical toxin profiles containing saxitoxin and gonyautoxins 2/3 isomers in the ratio of 1:9. This is the first evidence of paralytic shellfish toxins in this species and also the occurrence of the toxin producing cyanobacterium in South American countries.     

First evidence of “paralytic shellfish toxins” and cylindrospermopsin in a Mexican freshwater system, Lago Catemaco, and apparent bioaccumulation of the toxins in “tegogolo” snails (Pomacea patula catemacensis)

Exposure to cyanobacterial toxins in freshwater systems, including both direct (e.g., drinking water) and indirect (e.g., bioaccumulation in food webs) routes, is emerging as a potentially significant threat to human health. We investigated cyanobacterial toxins, specifically cylindrospermopsin (CYN), the microcystins (MCYST) and the “paralytic shellfish toxins” (PST), in Lago Catemaco (Veracruz, Mexico). Lago Catemaco is a tropical lake dominated by Cylindrospermopsis, specifically identified as Cylindrospermopsis catemaco and Cylindrospermopsis philippinensis, and characterized by an abundant, endemic species of snail (Pomacea patula catemacensis), known as “tegogolos,” that is both consumed locally and commercially important. Samples of water, including dissolved and particulate fractions, as well as extracts of tegogolos, were screened using highly specific and sensitive ELISA. ELISA identified CYN and PST at low concentrations in only one sample of seston; however, both toxins were detected at appreciable quantities in tegogolos. Calculated bioaccumulation factors (BAF) support bioaccumulation of both toxins in tegogolos. The presence of CYN in the phytoplankton was further confirmed by HPLC-UV and LC-MS, following concentration and extraction of algal cells, but the toxin could not be confirmed by these methods in tegogolos. These data represent the first published evidence for CYN and the PST in Lago Catemaco and, indeed, for any freshwater system in Mexico. Identification of the apparent bioaccumulation of these toxins in tegogolos may suggest the need to further our understanding of the transfer of cyanobacterial toxins in freshwater food webs as it relates to human health.     

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYP020B were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii.     

Effects of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum (cyanobacteria) ingestion on Daphnia magna midgut and associated diverticula epithelium

This article reports a light and electron microscopy investigation of the effects of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum ingestion on midgut and associated digestive diverticula of Daphnia magna. Additionally, survivorship and growth effects caused by feeding on cyanobacteria were assessed. Three cyanobacteria were used in the experiments: cylindrospermopsin (CYN)-producing C. raciborskii, CYN-producing A. ovalisporum and non-CYN-producing C. raciborskii. In order to discriminate between the alterations due to the low nutritional value of cyanobacteria and toxic effects, a control group was fed on the chlorophyte Ankistrodesmus falcatus and another control group was not fed. In the chlorophyte fed control, the epithelium lining the midgut and associated diverticula is mainly formed by strongly stained cells with an apical microvilli border. Nevertheless, unstained areas in which cell lyses had occurred were also observed. In the unfed control, the unstained areas became predominant due to an increment of cell lyses. All individuals fed on CYN-producing A. ovalisporum and some of those fed on non-CYN-producing C. raciborskii appear similar to the unfed control. However, some individuals fed on non-CYN-producing C. raciborskii showed similarities with the fed control. In contrast, the midgut and digestive diverticula of D. magna fed on CYN-producing C. raciborskii showed a widespread dissociation of epithelial cells, associated with severe intracellular disorganization, but cell lysis was less evident than in controls. These alterations cannot be attributed to CYN, because those effects were not induced by CYN-producing A. ovalisporum. Therefore, data suggest the production of another unidentified active metabolite by CYN-producing C. raciborskii, responsible for the disruption of cell adhesion in the epithelium of D. magna digestive tract. Data also show that the tested cyanobacteria are inadequate as food to D. magna, due to low nutritional value and toxic content.     

Cytotoxic and morphological effects of microcystin‐LR,cylindrospermopsin, and their combinations on the human hepatic cell line HepG2

Cylindrospermopsin (CYN) and Microcystin‐LR (MC‐LR) are toxins produced by different cyanobacterial species, which are found mainly in freshwater reservoirs. Both of them can induce, separately, toxic effects in humans and wildlife. However, little is known about the toxic effects of the combined exposure, which could likely happen, taking into account the concomitant occurrence of the producers. As both cyanotoxins are well known to induce hepatic damage, the human hepatocellular HepG2 cell line was selected for the present study. Thus, the cytotoxicity of both pure cyanotoxins alone (0–5 μg/mL CYN and 0–120 μg/mL MC‐LR) and in combination for 24 and 48 h was assayed, as long as the cytotoxicity of extracts from CYN‐producing and nonproducing cyanobacterial species. The potential interaction of the combination was evaluated by the isobologram or Chou–Talalay's method, which provides a combination index as a quantitative measure of the two cyanotoxins interaction's degree. Moreover, a morphological study of the individual pure toxins and their combinations was also performed. Results showed that CYN was the most toxic pure cyanotoxin, being the mean effective concentrations obtained ≈4 and 90 μg/mL for CYN and MC‐LR, respectively after 24 h. However, the simultaneous exposure showed an antagonistic effect. Morphologically, autophagy, at low concentrations, and apoptosis, at high concentrations were observed, with affectation of the rough endoplasmic reticulum and mitochondria. These effects were more pronounced with the combination. Therefore, it is important to assess the toxicological profile of cyanotoxins combinations in order to perform more realistic risk evaluations.