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.

Variability in retinoid-like activity of extracellular compound mixtures produced by wide spectra of phytoplankton species and contributing metabolites.

Some phytoplankton species were shown to produce teratogenic retinoids. This study assessed the variability in the extracellular production of compounds with retinoid-like activity for 50 independent cultivations of wide spectra of species including 12 cyanobacteria (15 strains) and 4 algae of different orders. Extracellular retinoid-like activity was detected for repeated cultivations of six cyanobacteria. The results were consistent for some species including Microcystis aeruginosa and Aphanizomenon gracile. The detected retinoid-like activities ranged from below the limit of quantification of 16 ng/L to over 6 µg all-trans retinoic acid (ATRA) equivalent/L. Nontargeted virtual fractionation together with suspect screening approach enabled to identify some retinoid-like compounds in exudates, including ATRA, 9/13-cis retinoic acid, all-trans 5,6-epoxy retinoic acid, 4keto-ATRA, 4keto-retinal, 4hydroxy-ATRA, and retinal. Most of them were for the first time repeatedly detected in exudates of all studied algae (at ng/L levels) and cyanobacteria. Their relative potencies ranged from 0.018 (retinal) to 1 compared to ATRA. They accounted for less than 0.1-50% of total detected retinoid-like activity. The high detected activities and concentrations of retinoids in some samples and their direct accessibility from exudates document potential risk of developmental toxicity for organisms in proximity of massive water blooms.

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.