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Retinoic acids and their derivatives have been recently identified by chemical analyses in cyanobacteria and algae. Given the essential role of retinoids for vertebrate development this has raised concerns about a potential risk for vertebrates exposed to retinoids during cyanobacterial blooms. Our study focuses on extracellular compounds produced by phytoplankton cells (exudates). In order to address the capacity for the production of retinoids or compounds with retinoid-like activity we compared the exudates of ten cyanobacteria and algae using in vitro reporter gene assay. Exudates of three cyanobacterial species showed retinoid-like activity in the range of 269-2,265 ng retinoid equivalents (REQ)/L, while there was no detectable activity in exudates of the investigated algal species. The exudates of one green alga (Desmodesmus quadricaudus) and the two cyanobacterial species with greatest REQ levels, Microcystis aeruginosa and Cylindrospermopsis raciborskii, were selected for testing of the potential relation of retinoid-like activity to developmental toxicity in zebrafish embryos. The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, spine and mouth deformation) and interference with growth in zebrafish embryos, while such effects were not observed for the alga. Fish embryos were also exposed to all-trans retinoic acid (ATRA) in a range equivalent to the REQ concentrations detected in exudates by in vitro bioassays. Both the phenotypes and effective concentrations of exudates corresponded to ATRA equivalents, supporting the hypothesis that the teratogenic effects of cyanobacterial exudates are likely to be associated with retinoid-like activity. The study documents that some cyanobacteria are able to produce and release retinoid-like compounds into the environment at concentrations equivalent to those causing teratogenicity in zebrafish. Hence, the characterization of retinoid-like and teratogenic potency should be included in the assessment of the potential adverse effects caused by the release of toxic and bioactive compounds during cyanobacterial blooms.

The biological activity of cyanobacteria and their chemical components have been widely studied due to their blooms in eutrophic waters worldwide. The primary goal of this study was to determine if individual cyanobacterial species and mixtures of cyanobacteria collected from the environment contain compounds with the potential for interaction with signaling pathways of the aryl hydrocarbon receptor (AhR), androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR) and retinoid acid receptor (RAR). Cytotoxicity and specific toxic potencies of products of freshwater cyanobacteria were determined by use of in vitro reporter gene trans-activation assays. The testing included samples prepared from five selected single cyanobacterial species cultivated in laboratory and five complex cyanobacterial biomasses collected from blooms in surface waters in the Czech Republic. The results demonstrate estrogenic potencies of extracts of cyanobacterial biomasses. Among the laboratory single species, the extract of Planktothrix agardhii (intracellular metabolites) had a potency of estrogenic equivalents (EEQ) of 3.8 ng 17β-estradiol/g dw. The estimates of EEQs of samples prepared from complex cyanobacterial biomasses collected from freshwaters in the Czech Republic ranged from 19 to 2200 ng 17β-estradiol/g dw. Several samples prepared from the environmental cyanobacterial biomasses potentiated the androgenic potency of dihydrotestosterone. There was no dioxin-like, glucocorticoid or anti/retinoic activity observed for any of the extracts studied. Extracts of natural complex cyanobacterial biomasses exhibited greater and more frequent presence of compounds with specific modes of action, mainly estrogenic, and also greater cytotoxicity than extracts of single cyanobacterial species. The demonstrated estrogenic potency of the compounds present in complex cyanobacterial biomasses is of environmental relevance, and could potentially contribute to endocrine disruptive effects in aquatic ecosystems in case of great bloom densities.

Lake Pilnok located in the black coal-mining region Ostrava-Karvina, Czech Republic, contains sediments highly contaminated with powdered waste coal. Moreover, population of the endangered species of narrow-clawed crayfish Pontastacus leptodactylus with high proportion of intersex individuals (18%) was observed at this site. These findings motivated our work that aimed to evaluate contamination, endocrine disruptive potency using in vitro assays and in vivo effects of contaminated sediments on reproduction of sediment-dwelling invertebrates. Chemical analyses revealed low concentrations of persistent chlorinated compounds and heavy metals but concentrations of polycyclic aromatic hydrocarbons (PAH) were high (sum of 16 PAHs 10g/g dw). Organic extracts from sediments caused significant in vitro AhR-mediated activity in the bioassay with H4IIE-luc cells, estrogenicity in MVLN cells and anti-androgenicity in recombinant yeast assay, and these effects could be attributed to non-persistent compounds derived from the waste coal. We have also observed significant in vivo effects of the sediments in laboratory experiments with the Prosobranchian euryhaline mud snail Potamopyrgus antipodarum. Sediments from Lake Pilnok as well as organic extracts of the sediments (externally added to the control sediment) significantly affected fecundity during 8 weeks of exposure. The effects were stimulations of fecundity at lower concentrations at the beginning of the experiment followed by inhibitions of fecundity and general toxicity. Our study indicates presence of chemicals that affected endocrine balance in invertebrates, and emphasizes the need for integrated approaches combining in vitro and in vivo bioassays with identification of chemicals to elucidate ecotoxicogical impacts of contaminated sediment samples. © 2008 Elsevier B.V. All rights reserved.

While numerous studies focused on the effects of microcystins, the role of other components of complex cyanobacterial water blooms in toxicity is poorly understood. In this study we have evaluated effects of various fractions of cyanobacterial biomass with different composition and microcystin content on embryolarval development of carp (Cyprinus carpio). The following samples (fractions) of four natural water blooms were prepared and tested: complex cyanobacterial biomass, crude aqueous extract of biomass, cellular pellet remaining from aqueous extract, permeate (i.e. microcystin-free fraction prepared during C-18 solid-phase extraction; SPE), and eluate (i.e. fraction prepared by SPE containing mostly microcystins). Complex biomass and the crude aqueous extract (regardless of microcystin content and/or microcystin variants present) in the sample were the most toxic. On the other hand, eluate fractions of all samples containing microcystins in concentrations 8–255 gL −1 induced no or only weak toxic effects. Exposures of fish to permeate fractions (with removed microcystins) of two samples dominated by Aphanizomenon sp. and Planktothrix sp. resulted in significant mortality, while other two samples dominated by Microcystis spp. induced minor effects. We have also observed significant inhibition of glutathione S-transferases (GST) at most fractions of the Aphanizomenon sp. and Planktothrix sp. dominated samples. Our data indicate that cyanobacterial water blooms as well complex biomass extracts induce significant embryolarval toxicity in common carp. However, these effects were independent of microcystin content, and the most pronounced effects were observed with the non-Microcystis dominated samples. Therefore, a critical examination of microcystin role in overal ecotoxicology of complex cyanobacterial blooms is needed. © 2007 Elsevier B.V. All rights reserved.