Potential risk of estrogenic compounds produced by water blooms to aquatic environment.

Some freshwater phytoplankton species have been suggested to produce estrogenic compounds in concentrations which could cause adverse effects to aquatic biota, while other studies showed no estrogenic effects after exposure to phytoplankton extracts or pointed out possible sources of the overestimation of the estrogenic activity. This study aimed to clarify these research inconsistencies by investigating estrogenicity of biomass extracts from both environmental freshwater blooms and laboratory cyanobacterial and algae cultures by in vitro reporter bioassay. Biomasses of 8 cyanobacterial and 3 algal species from 7 taxonomic orders were extracted and tested. Next to this, samples of environmental water blooms collected from 8 independent water bodies dominated by phytoplankton species previously assessed as laboratory cultures were tested. The results showed undetectable or low estrogenicity of both freshwater blooms and laboratory cultures with E2 equivalent concentration (EEQ) in a range from LOQ up to 4.5 ng EEQ/g of dry mass. Moreover, the co-exposure of biomass extracts with environmentally relevant concentration of model estrogen (steroid hormone 17ß-estradiol; E2), commonly occurring in surface waters, showed simple additive interaction. However, some of the biomass extracts elicited partially anti-estrogenic effects in co-exposure with higher E2 concentration. In conclusion, our study documents undetectable or relatively low estrogenic potential of biomass extracts from both environmental freshwater blooms and studied laboratory cultured cyanobacterial and algae species. Nevertheless, in case of very high-density water blooms, even this low estrogenicity (detected for two cyanobacterial species) could lead to EEQ content in biomass reaching effect-based trigger values indicating potential risk, if recalculated per water volume at field sites. However, these levels would not occur in water under realistic environmental scenarios and the potential estrogenic effects would be most probably minor compared to other toxic effects caused by massive freshwater blooms of such high densities.

Muddying the unexplored post-industrial waters: Biodiversity and conservation potential of freshwater habitats in fly ash sedimentation lagoons.

Deposits of fly ash and other coal combustion wastes are common remnants of the energy industry. Despite their environmental risks from heavy metals and trace elements, they have been revealed as refuges for threatened terrestrial biodiversity. Surprisingly, freshwater biodiversity of fly ash sedimentation lagoons remains unknown despite such lack of knowledge strongly limits the efficient restoration of fly ash deposits. We bring the first comprehensive survey of freshwater biodiversity, including nekton, benthos, zooplankton, phytoplankton, and macrophytes, in fly ash lagoons across industrial regions of the Czech Republic. To assess their conservation potential, we compared their biodiversity with abandoned post-mining ponds, the known strongholds of endangered aquatic species in the region with a shortage of natural ponds. Of 28 recorded threatened species, 15 occurred in the studied fly ash lagoons, some of which were less abundant or even absent in the post-mining ponds. These are often species of nutrient-poor, fishless waters with rich vegetation, although some are specialised extremophiles. Species richness and conservation value of most groups in the fly ash lagoons did not significantly differ from the post-mining ponds, except for species richness of benthos, zooplankton, and macrophytes, which were slightly lower in the fly ash lagoons. Although the concentrations of some heavy metals (mainly Se, V, and As) were significantly higher in the fly ash lagoons, they did not significantly affect species richness or conservation value of the local communities. The differences in species composition therefore does not seem to be caused by water chemistry. Altogether, we have shown that fly ash lagoons are refuges for threatened aquatic species, and we thus suggest maintaining water bodies during site restoration after the cessation of fly ash deposition. Based on our analyses of environmental variables, we discuss suitable restoration practices that efficiently combine biodiversity protection and environmental risk reduction.

Estrogenic and retinoid-like activity in stagnant waters with mass occurrence of water blooms.

Stagnant freshwaters can be affected by anthropogenic pollution and eutrophication that leads to massive growth of cyanobacteria and microalgae forming complex water blooms. These can produce various types of bioactive compounds, some of which may cause embryotoxicity, teratogenicity, endocrine disruption and impair animal or human health. This study focused on potential co-occurrence of estrogenic and retinoid-like activities in diverse stagnant freshwaters affected by phytoplankton blooms with varying taxonomic composition. Samples of phytoplankton bloom biomass and its surrounding water were collected from 17 independent stagnant water bodies in the Czech Republic and Hungary. Total estrogenic equivalents (EEQ) of the most potent samples reached up to 4.9 ng·g-1 dry mass (dm) of biomass extract and 2.99 ng·L-1 in surrounding water. Retinoic acid equivalent (REQ) measured by in vitro assay reached up to 3043 ng·g-1 dm in phytoplankton biomass and 1202 ng·L-1in surrounding water. Retinoid-like and estrogenic activities at some sites exceeded their PNEC and effect-based trigger values, respectively. The observed effects were not associated with any particular species of cyanobacteria or algae dominating the water blooms nor related to phytoplankton density. We found that taxonomically diverse phytoplankton communities can produce and release retinoid-like compounds to surrounding water, while estrogenic potency is likely related to estrogens of anthropogenic origin adsorbed to phytoplankton biomass. Retinoids occurring in water blooms are ubiquitous signalling molecules, which can affect development and neurogenesis. Selected water bloom samples (both water and biomass extracts) with retinoid-like activity caused effects on neurodifferentiation in vitro corresponding to those of equivalent all-trans-retinoic acid concentrations. Co-occurrence of estrogenic and retinoid-like activities in stagnant water bodies as well as the potential of compounds produced by water blooms to interfere with neural differentiation should be considered in the assessment of risks associated with water blooms, which can comprise complex mixtures of natural and anthropogenic bioactive compounds.

Occurrence of cylindrospermopsin, anatoxin-a and their homologs in the southern Czech Republic - Taxonomical, analytical, and molecular approaches.

Water bloom-forming cyanobacteria have a severe impact on freshwater quality. Although some cyanobacterial toxins such as microcystins have been studied extensively, other toxins like anatoxin-a (ATX) and their structural analogs - as well as cyanobacterial taxa producing these toxins remain to be explored in detail. The present study investigated levels of ATX, CYN and their homologs along with the occurrence of anaC and cyrJ genes in water blooms in 16 sites in the Czech Republic that were pre-selected concerning the presence of potential toxin producers. Besides, we also studied toxins and genes in a series of strains available in our laboratories. ATX and its congener HATX were detected in 5 natural biomass samples from the Czech Republic (maximum concentration 2.8 micrograms per gram d.w.). Interestingly, the anaC gene coding for ATX production was not detected in any of these toxin-positive biomass samples. The concentrations of ATX congeners in cyanobacterial laboratory strains were about 10-times higher than those of the original ATX, which calls for further research addressing levels and hazards of ATX analogs. Regarding the CYN and 7-deoxyCYN (other CYN congeners were not analyzed in this study) - these toxins were identified in a single small pond in the Czech Republic at concentrations 4.3 and 2.7 micrograms per gram of biomass d.w., respectively (corresponded to dissolved concentrations higher than 1 microgram per liter). The CYN-positive sample was dominated by CYN-producing taxa Raphidiopsis (basionym Cylindrospermopsis) and Cuspidothrix. We also confirmed the presence of a specific cyrJ gene in this natural bloom sample. To our knowledge, this is the first study pointing to Raphidiopsis (Cylindrospermopsis) and Cuspidothrix as producers of CYN in Europe. This observation calls for further research because of their increasing occurrence in (Central) Europe along with the global change. The present study demonstrates the importance of using combined (taxonomical, analytical, and molecular) approaches in the assessment of hazardous cyanobacteria and their toxins in freshwaters.

Tenebriella gen. nov. - The dark twin of Oscillatoria.

Oscillatoria has long been known to be polyphyletic. After recent resequencing of the reference strain for this genus, many Oscillatoria-like groups phylogenetically distant from the type species O. princeps remained unresolved. Here we describe one of these groups as a new genus Tenebriella. Most of the studied strains originate from Central Europe, where they are able to form prominent microbial mats. Despite the overall Oscillatoria-like morphology, Tenebriella can be distinguished by darker trichomes and forms a separate monophyletic clade in phylogenies inferred from the 16S rRNA gene and two additional loci (rpoC1, rbcLX). Within Tenebriella we recognize two new species differing from each other by morphological and ecological characteristics. First species does not fit any known taxon description, and thus is described as a new species T. amphibia. The latter one corresponds with the information available for Oscillatoria curviceps Agardh ex Gomont, and thus new combination T. curviceps is proposed. The phylogenetic analyses of the 16S-23S ITS region together with the comparison of the hypothetical secondary structures confirmed recognition of these two species and additionally revealed presence of a morphologically cryptic species Tenebriella sp. The results corroborate frequent recurrence of convergent morphotypes in the evolution of cyanobacteria and justify further exploration even of the intensively studied European freshwaters using molecular phylogenetics to discover new and ecologically relevant taxa.

Widespread occurrence of retinoids in water bodies associated with cyanobacterial blooms dominated by diverse species.

Cyanobacterial blooms represent a worldwide problem in freshwater as well as marine ecosystems as producers of various toxic compounds. This study provides environmentally important information about the common presence of mixtures of retinoids in various water bodies associated with the occurrence of cyanobacterial blooms dominated by many different species. The study documents, for the first time, that retinoids are produced by environmental cyanobacterial blooms dominated by species belonging to different genera such as Microcystis, Dolichospermum, Planktothrix, Woronichinia, Pseudanabaena and others. Samples of biomass of cyanobacterial blooms and their surrounding water were collected from seventeen independent freshwater bodies across the Czech Republic during summer 2015. Retinoid-like activity was detected by an in vitro reporter gene bioassay in water samples from 8 out of 17 localities with a maximal activity of 263 ng all-trans retinoic acid equivalent (REQ)/L. In comparison, in vitro assessment of biomass extracts documented retinoid-like activity at 11 out of 17 localities with a maximal retinoid-like activity of 867 ng REQ/g dry mass (dm). Individual retinoids were detected by chemical analyses in all water samples and in 16 out of 17 biomass samples with 4keto-retinal and all-trans 5,6epoxy retinoic acid being detected in aquatic ecosystems for the first time. Further, all-trans 4keto retinoic acid and retinal were the most commonly detected compounds in both types of samples. With respect to retinoid-like activity, a large proportion was explained in some samples by contributions of individual detected retinoids calculated from their concentrations and relative potencies. However, results also indicate that other unknown compounds with a retinoic acid receptor-mediated mode of action were present. The revealed widespread production of retinoids by cyanobacterial blooms dominated by diverse species across various aquatic ecosystems and their common presence in both biomass and surrounding water raises concern namely because some retinoids belong to the most potent teratogens. These compounds need to be taken into consideration in the assessment of risks associated with massive cyanobacterial blooms.

A new microcystin producing Nostoc strain discovered in broad toxicological screening of non-planktic Nostocaceae (cyanobacteria).

Benthic cyanobacteria recognized as producers of natural products, including cyanotoxins, have been neglected for systematic toxicological studies. Thus, we have performed a broad study investigating cyanotoxin potential of 311 non-planktic nostocacean representatives combining molecular and chemical analyses. Out of these, a single strain Nostoc sp. Treb K1/5, was identified as a new microcystin producer. Microcystins [Asp3]MC-YR, [Asp3]MC-FR, [Asp3]MC-HtyR and Ala-Leu/Ile-Asp-Arg-Adda-Glu-Mdha are reported for the first time from the genus Nostoc. All the studied strains were also analyzed for the occurrence of nodularins, cylindrospermopsin and (homo)anatoxin-a, yet no novel producer has been discovered. Our findings indicate rare occurrence of the common cyanotoxins in non-planktic nostocaceae which is in contrast with frequent reports of cyanotoxin producers among phylogenetically closely related planktic cyanobacteria.

Phytoestrogens and sterols in waters with cyanobacterial blooms - Analytical methods and estrogenic potencies.

Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, ß-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 µg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17ß-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17ß-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17ß-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.

Assessment of Chemical Impact of Invasive Bryozoan Pectinatella magnifica on the Environment: Cytotoxicity and Antimicrobial Activity of P. magnifica Extracts.

Pectinatella magnifica, an invasive bryozoan, might significantly affect ecosystem balance due to its massive occurrence in many areas in Europe and other parts of the world. Biological and chemical analyses are needed to get complete information about the impact of the animal on the environment. In this paper, we aimed to evaluate in vitro cytotoxic effects of five extracts prepared from P. magnifica using LDH assay on THP-1 cell line. Antimicrobial activities of extracts against 22 different bacterial strains were tested by microdilution method. Our study showed that all extracts tested, except aqueous portion, demonstrated LD50 values below 100 µg/mL, which indicates potential toxicity. The water extract of P. magnifica with LD50 value of 250 µg/mL also shows potentially harmful effects. Also, an environmental risk resulting from the presence and increasing biomass of potentially toxic benthic cyanobacteria in old colonies should not be underestimated. Toxicity of Pectinatella extracts could be partially caused by presence of Aeromonas species in material, since we found members of these genera as most abundant bacteria associated with P. magnifica. Furthermore, P. magnifica seems to be a promising source of certain antimicrobial agents. Its methanolic extract, hexane, and chloroform fractions possessed selective inhibitory effect on some potential pathogens and food spoiling bacteria in the range of MIC 0.5-10 mg/mL. Future effort should be made to isolate and characterize the content compounds derived from P. magnifica, which could help to identify the substance(s) responsible for the toxic effects of P. magnifica extracts.