Global Metabolomic Characterizations of Microcystis spp. Highlights Clonal Diversity in Natural Bloom-Forming Populations and Expands Metabolite Structural Diversity.

Cyanobacteria are photosynthetic prokaryotes capable of synthesizing a large variety of secondary metabolites that exhibit significant bioactivity or toxicity. Microcystis constitutes one of the most common cyanobacterial genera, forming the intensive blooms that nowadays arise in freshwater ecosystems worldwide. Species in this genus can produce numerous cyanotoxins (i.e., toxic cyanobacterial metabolites), which can be harmful to human health and aquatic organisms. To better understand variations in cyanotoxin production between clones of Microcystis species, we investigated the diversity of 24 strains isolated from the same blooms or from different populations in various geographical areas. Strains were compared by genotyping with 16S-ITS fragment sequencing and metabolite chemotyping using LC ESI-qTOF mass spectrometry. While genotyping can help to discriminate among different species, the global metabolome analysis revealed clearly discriminating molecular profiles among strains. These profiles could be clustered primarily according to their global metabolite content, then according to their genotype, and finally according to their sampling location. A global molecular network of all metabolites produced by Microcystis species highlights the production of a wide set of chemically diverse metabolites, including a few microcystins, many aeruginosins, microginins, cyanopeptolins, and anabaenopeptins, together with a large set of unknown molecules. These components, which constitute the molecular biodiversity of Microcystis species, still need to be investigated in terms of their structure and potential bioactivites (e.g., toxicity).

Subcellular localization of microcystin in the liver and the gonads of medaka fish acutely exposed to microcystin-LR.

Among the diverse toxic components produced by cyanobacteria, microcystins (MCs) are one of the most toxic and notorious cyanotoxin groups. Besides their potent hepatotoxicity, MCs have been revealed to induce potential reproductive toxicity in various animal studies. However, little is still known regarding the distribution of MCs in the reproductive organ, which could directly affect reproductive cells. In order to respond to this question, an acute study was conducted in adult medaka fish (model animal) gavaged with 10 µg.g-1 body weight of pure MC-LR. The histological and immunohistochemical examinations reveal an intense distribution of MC-LR within hepatocytes along with a severe liver lesion in the toxin-treated female and male fish. Besides being accumulated in the hepatocytes, MC-LR was also found in the connective tissue of the ovary and the testis, as well as in oocytes and degenerative spermatocyte-like structures but not spermatocytes. Both liver and gonad play important roles in the reproductive process of oviparous vertebrates. This observation constitutes the first observation of the presence of MC-LR in reproductive cells (female, oocytes) of a vertebrate model with in vivo study. Our results, which provide intracellular localization of MC-LR in the gonad, advance our understanding of the potential reproductive toxicity of MC-LR in fish.

Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver.

Cyanobacterial blooms have become a common phenomenon in eutrophic freshwater ecosystems worldwide. Microcystis is an important bloom-forming and toxin-producing genus in continental aquatic ecosystems, which poses a potential risk to Human populations as well as on aquatic organisms. Microcystis is known to produce along with various bioactive peptides, the microcystins (MCs) that have attracted more attention notably due to their high hepatotoxicity. To better understand the effects of cyanobacterial blooms on fish, medaka fish (Oryzias latipes) were sub-chronically exposed to either non-MC-producing or MC-producing living strains and, for this latter, to its subsequent MC-extract of Microcystis aeruginosa. Toxicological effects on liver have been evaluated through the combined approach of histopathology and 'omics' (i.e. proteomics and metabolomics). All treatments induce sex-dependent effects at both cellular and molecular levels. Moreover, the modalities of exposure appear to induce differential responses as the direct exposure to the cyanobacterial strains induce more acute effects than the MC-extract treatment. Our histopathological observations indicate that both non-MC-producing and MC-producing strains induce cellular impairments. Both proteomic and metabolomic analyses exhibit various biological disruptions in the liver of females and males exposed to strain and extract treatments. These results support the hypothesis that M. aeruginosa is able to produce bioactive peptides, other than MCs, which can induce toxicological effects in fish liver. Moreover, they highlight the importance of considering cyanobacterial cells as a whole to assess the realistic environmental risk of cyanobacteria on fish.

An integrated omic analysis of hepatic alteration in medaka fish chronically exposed to cyanotoxins with possible mechanisms of reproductive toxicity.

Cyanobacterial blooms threaten human health as well as the population of other living organisms in the aquatic environment, particularly due to the production of natural toxic components, the cyanotoxin. So far, the most studied cyanotoxins are microcystins (MCs). In this study, the hepatic alterations at histological, proteome and transcriptome levels were evaluated in female and male medaka fish chronically exposed to 1 and 5 µg L-1 microcystin-LR (MC-LR) and to the extract of MC-producing Microcystis aeruginosa PCC 7820 (5 µg L-1 of equivalent MC-LR) by balneation for 28 days, aiming at enhancing our understanding of the potential reproductive toxicity of cyanotoxins in aquatic vertebrate models. Indeed, both MC and Microcystis extract adversely affect reproductive parameters including fecundity and egg hatchability. The liver of toxin treated female fish present glycogen storage loss and cellular damages. The quantitative proteomics analysis revealed that the quantities of 225 hepatic proteins are dysregulated. In particular, a notable decrease in protein quantities of vitellogenin and choriogenin was observed, which could explain the decrease in reproductive output. Liver transcriptome analysis through Illumina RNA-seq reveals that over 100-400 genes are differentially expressed under 5 µg L-1 MC-LR and Microcystis extract treatments, respectively. Ingenuity pathway analysis of the omic data attests that various metabolic pathways, such as energy production, protein biosynthesis and lipid metabolism, are disturbed by both MC-LR and the Microcystis extract, which could provoke the observed reproductive impairment. The transcriptomics analysis also constitutes the first report of the impairment of circadian rhythm-related gene induced by MCs. This study contributes to a better understanding of the potential consequences of chronic exposure of fish to environmental concentrations of cyanotoxins, suggesting that Microcystis extract could impact a wider range of biological pathways, compared with pure MC-LR, and even 1 µg L-1 MC-LR potentially induces a health risk for aquatic organisms.

Deep sexual dimorphism in adult medaka fish liver highlighted by multi-omic approach.

Sexual dimorphism describes the features that discriminate between the two sexes at various biological levels. Especially, during the reproductive phase, the liver is one of the most sexually dimorphic organs, because of different metabolic demands between the two sexes. The liver is a key organ that plays fundamental roles in various physiological processes, including digestion, energetic metabolism, xenobiotic detoxification, biosynthesis of serum proteins, and also in endocrine or immune response. The sex-dimorphism of the liver is particularly obvious in oviparous animals, as the female liver is the main organ for the synthesis of oocyte constituents. In this work, we are interested in identifying molecular sexual dimorphism in the liver of adult medaka fish and their sex-variation in response to hepatotoxic exposures. By developing an integrative approach combining histology and different high-throughput omic investigations (metabolomics, proteomics and transcriptomics), we were able to globally depict the strong sexual dimorphism that concerns various cellular and molecular processes of hepatocytes comprising protein synthesis, amino acid, lipid and polysaccharide metabolism, along with steroidogenesis and detoxification. The results of this work imply noticeable repercussions on the biology of oviparous organisms environmentally exposed to chemical or toxin issues.

Gender-Specific Toxicological Effects of Chronic Exposure to Pure Microcystin-LR or Complex Microcystis aeruginosa Extracts on Adult Medaka Fish.

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to other organisms. However, their overall and specific implications for the health of aquatic organisms that are chronically and environmentally exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with other bioactive compounds have still not been clearly established and remain difficult to assess. The medaka fish was chosen as the experimental aquatic model for studying the cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing or nonproducing cyanobacterial biomasses, and of a Microcystis aeruginosa natural bloom. Our results showed a higher susceptibility of females to the different treatments compared to males at both the cellular and the molecular levels. Although hepatocyte lysis increased with MC-containing treatments, lysis always appeared more severe in the liver of females compare to males, and the glycogen cellular reserves also appeared to decrease more in the liver of females compared to those in the males. Proteomic investigations reveal divergent responses between males and females exposed to all treatments, especially for proteins involved in metabolic and homeostasis processes. Our observations also highlighted the dysregulation of proteins involved in oogenesis in female livers. These results suggest that fish populations exposed to cyanobacteria blooms may potentially face several ecotoxicological issues.

Detection of free and covalently bound microcystins in different tissues (liver, intestines, gills, and muscles) of rainbow trout (Oncorhynchus mykiss) by liquid chromatography-tandem mass spectrometry: method characterization.

So far only a few publications have explored the development of extraction methods of cyanotoxin extracted from complex matrices. With regard to cyanobacterial microcystins (MCs), the data on the contamination of the flesh of aquatic organisms is hard to compare and very limited due to the lack of validated methods. In recent years, evidence that both free and bound fractions of toxin are found in these tissues has highlighted the need to develop effective methods of quantification. Several techniques do exist, but only the Lemieux oxidation has so far been used to investigate complex tissue matrices. In this study, protocols based on the Lemieux approach were adapted for the quantitative chemical analysis of free MC-LR and MMPB derived from bound toxin in the tissues of juvenile trout gavaged with MC-LR. Afterwards, the NF V03 110 guideline was used to characterize the protocols elaborated and evaluate their effectiveness.

Toxicity of harmful cyanobacterial blooms to bream and roach.

Aquatic ecosystems are facing increasing environmental pressures, leading to an increasing frequency of cyanobacterial Harmful Algal Blooms (cHABs) that have emerged as a worldwide concern due to their growing frequency and their potential toxicity to the fauna that threatens the functioning of ecosystems. Cyanobacterial blooms raise concerns due to the fact that several strains produce potent bioactive or toxic secondary metabolites, such as the microcystins (MCs), which are hepatotoxic to vertebrates. These strains of cyanobacteria may be potentially toxic to fish via gastrointestinal ingestion and also by direct absorption of the toxin MC from the water. The purpose of our study was to investigate toxic effects observed in fish taken from several lakes in the Ile-de-France region, where MCs-producing blooms occur. This study comprises histological studies and the measurement of MC concentrations in various organs. The histological findings are similar to those obtained following laboratory exposure of medaka fish to MCs: hepatic lesions predominate and include cell lysis and cell detachment. MC concentrations in the organs revealed that accumulation was particularly high in the digestive tract and the liver, which are known to be classical targets of MCs. In contrast concentrations were very low in the muscles. Differences in the accumulation of MC variants produced by blooms indicate that in order to more precisely evaluate the toxic potential of a specific bloom it is necessary not only to consider the concentration of toxins, but also the variants produced.

Marked toxicity of Albizia bernieri extracts on embryo-larval development in the medaka fish (Oryzias latipes).

Previous phytochemical studies have shown that the plants of the Albizia genus (Fabaceae) contain bioactive saponins, lignans, spermine alkaloids, flavonoids, glycosides phenols and pyridoxine derivatives. Their extracts sometimes display medical properties, but can have also toxic effects. The purpose of our study was to determine the in vivo toxicity of Albizia bernieri seeds in the experimental model of the medaka fish embryo, which is recommended for use in toxicity studies. Our results show clearly that incubating the embryos or larvae of the medaka fish in a medium containing A. bernieri extracts caused a dose-dependent reduction in embryo or larvae survival. Embryos exposed to an extract of A. bernieri displayed cerebral lesions, such as cell lysis and the emergence of lysosomes in the glial tissue. We conclude that when comparing with data obtained with different plant extracts tested on medaka development in our laboratory, A. bernieri displays an unusually high toxicity. Focussing on the cerebral target as well as the fish behaviour could bring more specific informations.

Effects of a toxic cyanobacterial bloom (Planktothrix agardhii) on fish: insights from histopathological and quantitative proteomic assessments following the oral exposure of medaka fish (Oryzias latipes).

Cyanobacterial toxic blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to fish and other aquatic organisms. Microcystin-LR (the cyanotoxin most commonly detected in the freshwater environment) is a potent hepatotoxin, deregulating the kinase pathway by inhibiting phosphatases 1 and 2A. Although toxicological effects have been clearly linked to the in vitro exposure of fish to purified microcystins, cyanotoxins are produced by the cyanobacteria together with numerous other potentially toxic molecules, and their overall and specific implications for the health of fish have still not been clearly established and remain puzzlingly difficult to assess. The medaka fish (Oryzias latipes) was chosen as an in vitro model for studying the effects of a cyanobacterial bloom on liver protein contents using a gel free quantitative approach, iTRAQ, in addition to pathology examinations on histological preparations. Fish were gavaged with 5 µL cyanobacterial extracts (Planktothrix agardhii) from a natural bloom (La Grande Paroisse, France) containing 2.5 µg equiv. MC-LR. 2h after exposure, the fish were sacrificed and livers were collected for analysis. Histological observations indicate that hepatocytes present glycogen storage loss, and cellular damages, together with immunological localization of MCs. Using a proteomic approach, 304 proteins were identified in the fish livers, 147 of them with a high degree of identification confidence. Fifteen of these proteins were statistically significantly different from those of controls (gavaged with water only). Overall, these protein regulation discrepancies clearly indicate that oxidative stress and lipid regulation had occurred in the livers of the exposed medaka fish. In contrast to previous pure microcystin-LR gavage experiments, marked induction of vitellogenin 1 protein was observed for the first time with a cyanobacterial extract. This finding was confirmed by ELISA quantification of vitellogenin liver content, suggesting that the Planktothrix bloom extract had induced the occurrence of an endocrine-disrupting effect.

Pathological modifications following sub-chronic exposure of medaka fish (Oryzias latipes) to microcystin-LR.

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. MCs, especially MC-LR, cause toxic effects in animals and are a recognized potent cause of environmental stress and health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. Consequently, one of the major problems is the chronic exposure of fish to cyanotoxins in their natural environment. The present experiment involving chronic exposure confirmed initial findings on acute exposure to MC contamination: exacerbated physiological stress and tissue damage in several tissues of exposed medaka fish. The gonads were affected specifically. In female gonads the modifications included reduction of the vitellus storage, lysis of the gonadosomatic tissue and disruption of the relationships between the follicular cells and the oocytes. In the males, spermatogenesis appeared to be disrupted. This is the first report showing that a cyanotoxin can affect reproductive function, and so can impact on fish reproduction and thus fish stocks.

Oral toxicity of extracts of the microcystin-containing cyanobacterium Planktothrix agardhii to the medaka fish (Oryzias latipes).

As previously demonstrated the medaka fish appears to offer a good model for studies of microcystins (MCs) effects. Since cyanobacterial toxins are released with other molecules in the aquatic environment when the producers are dying, in this study, we performed additional experiments in order to compare the described effects obtained with the pure toxin microcystin-LR (MC-LR), among the most toxic MCs, to those induced by complex extracts of an MCs-producer Planktothrix agardhii, strain PMC 75.02 and a natural bloom containing the MCs-producer P. agardhii. The toxicity of these extracts containing several variants of MC was determined in adult medaka treated by gavage. Extracts of an MCs-free strain of P. agardhii (PMC 87.02) were assayed for comparison. Extracts effects were analysed on two tissues, liver and intestine by means of photon and transmission electron microscopy. MC was localized in these tissues by immunocytochemistry. No effect was detectable with extracts of the MCs-free P. agardhii strain. The two MCs-P. agardhii extracts (strain and natural bloom) were able to induce harmful effects in the liver and intestine of the medaka fish in acute intoxication by gavage. In these target organs as shown by toxin immunolocalization, reactions leading to cell disjunction and lysis were observed apparently associated with an immune reaction implying MC containing macrophages. These effects are similar to those previously described with photonic microscopy in medaka treated with pure MC-LR with additional results obtained under the electron microscope. Since no significant effect was detected with the MCs-free (PMC 87.02) extract, we then conclude that MCs, even in complex association with other cyanobacterial components, should be responsible for the toxic effects observed in treated fish.

Toxins and stress in fish: proteomic analyses and response network.

Fish models are increasingly used in toxicological studies in the laboratory as well as in the field. In addition to contributing to the analysis of toxicity mechanisms, one major aim is to select biomarkers from among the metabolic responses to toxic agents observed that could be useful for surveying the aquatic environment. Since proteomics is a developing field in toxicological research, it seems opportune to explore the data obtained using this approach. This article proposes an overview of proteomic studies of fish exposed to environmental stressors comprising a cyanotoxin and the response networks observed. We tend to take a broad view of how proteins communicate and function within the cell, often encompassing large numbers of proteins that operate in pathways. We start by presenting and discussing the data from four experiments in which the medaka fish was treated under the same conditions with the cyanotoxin, microcystin-LR (MC-LR). Liver proteins were analyzed using two techniques: 2D electrophoresis and LCMSMS. In the second and main part of our paper, the proteomic data obtained from fish contaminated with chemicals, including those reported above concerning the medaka fish intoxicated with MC-LR, are considered in the round in order to identify fish responses to chemical stress. A tentative general overview of how groups of proteins work together depending on exposure and/or subcellular location is proposed, with the inclusion of MC-LR data obtained in mice for comparison.

iTRAQ-based proteomic study of the effects of microcystin-LR on medaka fish liver.

Microcystins are cyanotoxins that occur in ground water and thus pose a potential health risk. Microcystin-LR (microcystin-leucine-arginine) is a potent hepatotoxin, and is suspected of being a tumour promoter. Poisoning with this toxin causes several dysfunctions in hepatocytes by inhibiting protein phosphatases 1 and 2A, and notably produces oxidative stress, disrupts the cytoskeleton, and deregulates mitogen-activated protein kinase pathway. Medaka fish (Oryzias latipes) was chosen as a model for studying the effects of this cyanotoxin on liver proteins using a gel-free approach, iTRAQ. Fish were gavaged with microcystin-LR. Two hours later, 325 proteins could be identified by Scaffold Q+ and 32 proteins revealed statistically significant variations above a ∣0.2∣ threshold of log(2) ratio by comparison with control. These proteins are mostly involved in the translation and maturation of proteins, lipid metabolism and detoxification. Notably, apolipoproteins are deregulated which indicates a possible alteration of chylomicron-mediated transport.

Effects of Madagascar yam extracts, Dioscorea antaly, on embryo-larval development of medaka fish, Oryzias latipes.

The yams edible starchy tubers, are of cultural, economic and nutritional importance in tropical and subtropical regions. The present study concerns the analysis at different levels of Dioscorea antaly toxicity to medaka embryo-larval development. The incubation of medaka fish embryos in a medium containing Dioscorea antaly extract resulted in a dose dependent reduction in survival rate. Survival rates were reduced up to 100% with extract concentrations of 4mg mL(-1). The LD(50) was estimated to be 0.86mg mL(-1)Dioscorea antaly. Anatomopathological studies did not show any caustic effects, irritation to mouth, throat or intestinal tract in surviving embryos but rather an inflammatory reaction in the liver. The data presented in this paper thus extends the use of medaka embryos as a valuable model to analyze the effects of food toxins.

Localization of microcystin-LR in medaka fish tissues after cyanotoxin gavage.

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. Over 70 MCs have been successfully isolated and identified, of which MC-LR is the most commonly occurring toxin. Microcystins, especially MC-LR, cause toxic effects in mammals, birds and fish and are a recognized potent cause of environmental stress and pose a potential health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. They also constitute a public health threat to people via drinking water and food chains. The concentrations of MC-LR can be very low, even in fish displaying severely disrupted tissues, which makes it essential to devise selective and sensitive histochemical methods for identifying and localizing MC-LR in target organs, such as liver and intestine. The aim of the study reported here was to analyze the presence of MC-LR in contaminated fish tissues using immunohistochemical methods. The present experiment involving subacute exposure confirmed our initial hypothesis that subacute and acute exposure to microcystin contamination can exacerbate physiological stress, induce sustained pathological damage, and affect the immune response in exposed medaka fish.

Proteomic study of the effects of microcystin-LR on organelle and membrane proteins in medaka fish liver.

The microcystin-leucine-arginine toxin (MC-LR) is produced by cyanobacteria that sometimes bloom in water reservoirs. It targets the liver, thus posing potential health risks to human and animals. Microcystin inhibits the protein phosphatases PP1 and PP2A, leading to diverse cellular deregulation processes. A proteomic approach was applied to the medaka fish (Oryzias latipes) to obtain an overview of the effects of MC-LR on the liver. As membrane and organelle proteins are major structural and functional components of several cell signalling pathways, we decided to investigate here the membrane and organelle-enriched fractions from the livers of control and MC-LR treated medaka fish. Seventeen proteins were identified by proteomic analysis as being modulated in response to MC-LR treatment. This is the first time for eight of them to be reported as being involved in MC-LR effects: prohibitin, fumarylacetoacetase, protein disulfide isomerase A4 and A6, glucose regulated protein 78kDa, 40S ribosomal protein SA, cytochrome b5, and ATP synthase mitochondrial d subunit. These proteins are involved in protein maturation or in the response to oxidative stress highlighting the role of organelles in protein processing and the complex cooperation associated with oxidative stress.

Effects of Rhodocodon madagascariensis extracts on embryo-larval development of medaka fish, Oryzias latipes.

Rhodocodon madagascariensis, also named Urginea mascarenensis, is a malagasy plant belonging to the Hyacinthaceae family. As for the other members of the endemic malagasy genus Rhodocodon, the chemical and toxicological properties of this species have not yet been studied. The present study concerns the analysis of the toxicity of R. madagascariensis to medaka embryo-larval development. The incubation of medaka fish embryos or larvae in a medium containing R. madagascariensis extract resulted in a dose dependent reduction in development of embryos leading to lethality and a drastic reduction in survival rate of exposed larvae. Survival rates were reduced up to 100% with an extract concentration of 4 mg mL(-1). The LD(50) was estimated to be 1 mg mL(-1). Anatomopathological studies did show some neuro-embryonal modifications in the encephalic region. The data presented in this paper thus extends the use of medaka embryos as a valuable model to detect and analyse the effects of plant toxins.

Global quantitative analysis of protein phosphorylation status in fish exposed to microcystin.

The hepatotoxins, microcystins (MCs) are potent inhibitors of protein phosphatases PP1 and PP2A. These nonribosomal peptides are getting more and more attention because of their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria. Herein, we report a toxicological study conducted on aquatic animal models such as the medaka fish. To date, the detailed mechanisms underlying the toxicity of microcystins are unknown. MC-leucine-arginine (MC-LR) is the most toxic and the most commonly encountered variant of MCs in aquatic environment. It has been used for toxicological investigations on the liver of intoxicated medaka. We performed differential proteome analyses of MC-LR-treated and untreated medaka fish to investigate the mechanisms of establishment of early responses to the toxin. The identification of proteins involved in these early responses might constitute candidates of biomarkers of MC-LR exposure. Cytosolic proteins from livers of exposed or nonexposed medaka were resolved by 2D electrophoresis and detected using stains specific for phosphoproteins and for whole protein content. Overall, 15 spots were found to vary significantly on the proteomic 2D maps or on the phosphoproteomic 2D maps. Of these 15 proteins, only two could not be identified by mass spectrometry. Among the other proteins that were identified, phenylalanine hydroxylase and keratin 18 (type I) showed variations in phoshoryl content in agreement with inhibition of PP2A activity after exposure of the fish to MC-LR. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress, and apoptosis. The functional implications of responses to MC-LR exposure of these proteins are discussed. The methodology described in this report should be widely used to a number of tissues and organisms, thus helping in the search for biomarkers of MC-LR contamination.

Effects of cyanobacterial crude extracts from Planktothrix agardhii on embryo-larval development of medaka fish, Oryzias latipes.

Embryonic toxicity from exposure to microcystins, cyclic hepatotoxic heptapeptides from cyanobacteria, receives increasing attention as a public human health biohazard. Using a microinjection technology, we have introduced cyanobacterial extracts from Planktothrix agardhii directly into the vitellus of late neurula embryos (stage 19) of medaka (Oryzias latipes). Microinjection (2 nL) of P. agardhii PMC 75.02 extract containing microcystins (MC) resulted in a dose-dependent mortality of embryos. Survival rates were reduced up to 81% with extract concentrations of 10 mg mL(-1) (EC(50)=7.8 microg mL(-1)). On the other hand, injection of P. agardhii PMC 87.02 extract in which no microcystin could be detected resulted in much less embryonal toxicity (EC(50)=460 microg mL(-1)). In addition, advanced embryonic hatching processing was limited with PMC 75.02 crude extract and less obvious than had been described with pure MC-LR injections. In agreement with the known hepatotoxic effects of microcystin, embryos injected with PMC 75.02 extract consistently displayed hepatobiliary abnormalities. Loss of glycogen content of the hepatocytes and hepatic haemorrhage were evidenced in surviving post-hatching juveniles. Thus, the methodology presented in this paper should be a valuable tool to analyse the effects of crude extracts of cyanobacterial toxins on the development of aquatic vertebrate embryos.