Wastewater treatment plants (WWTPs) are the primary source of micropollutants in aquatic ecosystems. Many micropollutants tend to bind to sediments and persist until remobilizion by bioturbation or flood events. Advanced effluent treatment by ozonation has been proven to eliminate most micropollutants. The present study characterizes sediments' toxic potential regarding zebrafish embryo development, which highly complex nervous system is vulnerable to exposure to neurotoxic substances. Furthermore, behavioral changes can be induced even at low pollutant concentrations and do not cause acute toxicity. The study area includes stretches of the main waterbody, the Wurm River (sampling sites W1-W5), and its tributary the Haarbach River (sampling sites H1, and H2) in North-Rhine Westphalia, Germany. Both waterbodies serve as recipients of WWTPs' effluents. The effluent entering the Haarbach River is conventionally treated, while the Wurm River receives ozonated effluent from the Aachen-Soers WWTP. Seven sampling sites up- and downstream of the WWTPs were investigated in June of two subsequent years. The first sampling campaign in 2017 was characterized by prolonged dry weather. The second sampling campaign in 2018 occurred after prolonged rain events and the release of the rainwater overflow basin. Direct exposure of zebrafish embryos to native sediments using the sediment contact test represented an ecologically realistic scenario and showed no acute sublethal effects. Exposure of the zebrafish embryo to freeze-dried sediments representing the ecotoxicological status of sediments during flood events unfolded acute sublethal toxicity. Behavioral studies with zebrafish larvae were an essential part of environmental neurotoxicity testing. Zebrafish larvae exposed to sediments' concentrations causing no acute effects led to behavioral changes signalizing neurotoxic substances in sediments. Polyaromatic hydrocarbons, polychlorinated biphenyls, and nitroaromatic compounds were identified as potential toxicity drivers, whereby the rainwater overflow basin served as a possible source of pollution. Mixture toxicity, effect-directed analysis, and further sediment monitoring are needed.
- Klíčová slova
- Freeze-dried sediments, Rainwater overflow, Sediment remobilization, Toxicity drivers,
- MeSH
- chemické látky znečišťující vodu * analýza toxicita MeSH
- dánio pruhované MeSH
- ekosystém MeSH
- geologické sedimenty MeSH
- larva MeSH
- ozon * analýza MeSH
- počasí MeSH
- polychlorované bifenyly * analýza MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu * MeSH
- ozon * MeSH
- polychlorované bifenyly * MeSH
Retinoids are newly detected compounds in aquatic ecosystems associated with cyanobacterial water blooms. Their potential health risks are only scarcely described despite numerous detections of all-trans retinoic acid (ATRA) and its derivatives in the environment. Besides the known teratogen ATRA there is only little or no information about their potency and namely their effects in vivo. We characterize ATRA and 8 other retinoids reported to occur in the environment for their bioactivity and teratogenicity using four in vitro reporter gene assays and zebrafish (Danio rerio) embryotoxicity assay. Our results document the ability of these compounds to interfere with retinoid signalling and cause teratogenicity at environmentally relevant levels with EC50 values at nM (hundreds of ng/L) levels and teratogenic indexes ranging from 2.8 (9cis retinoic acid) to 15.8 (retinal). The relative potency of individual compounds for teratogenicity ranged from 0.059 (retinal) to 0.96 (5,6-epoxy ATRA) when compared to ATRA. An environmentally relevant mixture of retinoids was tested showing good predictability of teratogenicity from the in vitro activities and additive toxicity of the mixture. The high teratogenicity of the newly described compounds associated with cyanobacteria presents a concern for developmental stages due to high conservation of the retinoid signalling across vertebrates.
- Klíčová slova
- In vitro, RAR, Retinoids, Teratogenicity, Zebrafish,
- MeSH
- chemické látky znečišťující vodu * toxicita MeSH
- dánio pruhované genetika MeSH
- ekosystém MeSH
- Microcystis * MeSH
- retinoidy toxicita MeSH
- sinice * MeSH
- teratogeny toxicita MeSH
- tretinoin toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu * MeSH
- retinoidy MeSH
- teratogeny MeSH
- tretinoin MeSH
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.
- Klíčová slova
- Cyanobacteria, Exudates, Retinoic acid receptor, Retinoid-like activity, Retinoids,
- MeSH
- Aphanizomenon * MeSH
- fytoplankton MeSH
- Microcystis * MeSH
- retinoidy MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- retinoidy MeSH
There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.
- Klíčová slova
- estrogen, fathead minnow, histology, proteomics, toxicity pathway, transcriptomics,
- MeSH
- chemické látky znečišťující vodu * toxicita MeSH
- Cyprinidae * MeSH
- ethinylestradiol toxicita MeSH
- proteomika MeSH
- sexuální diferenciace MeSH
- vitelogeniny MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chemické látky znečišťující vodu * MeSH
- ethinylestradiol MeSH
- vitelogeniny MeSH
Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L-1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.
- Klíčová slova
- ATRA, Biomarkers, Cyanobacteria, Locomotor response, Retinoids, Zebrafish,
- MeSH
- biomasa MeSH
- biotest MeSH
- chemické látky znečišťující vodu metabolismus toxicita MeSH
- chování zvířat účinky léků MeSH
- dánio pruhované růst a vývoj metabolismus MeSH
- embryo nesavčí účinky léků metabolismus MeSH
- exprese genu účinky léků MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- receptory kyseliny retinové genetika metabolismus MeSH
- sinice růst a vývoj metabolismus MeSH
- tretinoin metabolismus toxicita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu MeSH
- receptory kyseliny retinové MeSH
- tretinoin MeSH
Herbicides and their metabolites are often detected in water bodies where they may cause adverse effects to non-target organisms. Their effects at environmentally relevant concentrations are often unclear, especially concerning mixtures of pesticides. This study thus investigated the impacts of one of the most used herbicides: S-metolachlor and its two metabolites, metolachlor oxanilic acid (MOA) and metolachlor ethanesulfonic acid (MESA) on the development of zebrafish embryos (Danio rerio). Embryos were exposed to the individual substances and their environmentally relevant mixture until 120 hpf (hours post-fertilization). The focus was set on sublethal endpoints such as malformations, hatching success, length of fish larvae, spontaneous movements, heart rate and locomotion. Moreover, expression levels of eight genes linked to the thyroid system disruption, oxidative stress defense, mitochondrial metabolism, regulation of cell cycle and retinoic acid (RA) signaling pathway were analyzed. Exposure to S-metolachlor (1 μg/L) and the pesticide mixture (1 μg/L of each substance) significantly reduced spontaneous tail movements of 21 hpf embryos. Few rare developmental malformations were observed, but only in larvae exposed to more than 100 μg/L of individual substances (craniofacial deformation, non-inflated gas bladder, yolk sac malabsorption) and to 30 μg/L of each substance in the pesticide mixture (spine deformation). No effect on hatching success, length of larvae, heart rate or larvae locomotion were found. Strong responses were detected at the molecular level including induction of p53 gene regulating the cell cycle (the pesticide mixture - 1 μg/L of each substance; MESA 30 μg/L; and MOA 100 μg/L), as induction of cyp26a1 gene encoding cytochrome P450 (pesticide mixture - 1 μg/L of each substance). Genes implicated in the thyroid system regulation (dio2, thra, thrb) were all overexpressed by the environmentally relevant concentrations of the pesticide mixture (1 μg/L of each substance) and MESA metabolite (1 μg/L). Zebrafish thyroid system disruption was revealed by the overexpressed genes, as well as by some related developmental malformations (mainly gas bladder and yolk sac abnormalities), and reduced spontaneous tail movements. Thus, the thyroid system disruption represents a likely hypothesis behind the effects caused by the low environmental concentrations of S-metolachlor, its two metabolites and their mixture.
- Klíčová slova
- Embryotoxicity, Environmental concentration, Metabolite, Pesticide mixture, Sublethal effects, Zebrafish embryo,
- MeSH
- acetamidy metabolismus toxicita MeSH
- chemické látky znečišťující vodu metabolismus toxicita MeSH
- dánio pruhované metabolismus MeSH
- embryo nesavčí účinky léků metabolismus MeSH
- embryonální vývoj účinky léků MeSH
- herbicidy metabolismus toxicita MeSH
- larva MeSH
- štítná žláza účinky léků embryologie MeSH
- synergismus léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetamidy MeSH
- chemické látky znečišťující vodu MeSH
- herbicidy MeSH
- metolachlor MeSH Prohlížeč
Cyanobacteria routinely release potentially harmful bioactive compounds into the aquatic environment. Several recent studies suggested a potential link between the teratogenicity of effects caused by cyanobacteria and production of retinoids. To investigate this relationship, we analysed the teratogenicity of field-collected cyanobacterial bloom samples by means of an in vivo zebrafish embryo test, an in vitro reporter gene bioassay and by the chemical analysis of retinoids. Extracts of biomass from cyanobacterial blooms with the dominance of Microcystis aeruginosa and Aphanizomenon klebahnii were collected from water bodies in the Czech Republic and showed significant retinoid-like activity in vitro, as well as high degrees of teratogenicity in vivo. Chemical analysis was then used to identify a set of retinoids in ng per gram of dry weight concentration range. Subsequent fractionation and bioassay-based characterization identified two fractions with significant in vitro retinoid-like activity. Moreover, in most of the retinoids eluted from these fractions, teratogenicity with malformations typical for retinoid signalling disruption was observed in zebrafish embryos after exposure to the total extracts and these in vitro effective fractions. The zebrafish embryo test proved to be a sensitive toxicity indicator of the biomass extracts, as the teratogenic effects occurred at even lower concentrations than those expected from the activity detected in vitro. In fact, teratogenicity with retinoid-like activity was detected at concentrations that are commonly found in biomasses and even in bulk water surrounding cyanobacterial blooms. Overall, these results provide evidence of a link between retinoid-like activity, teratogenicity and the retinoids produced by cyanobacterial water blooms in the surrounding environment.
- Klíčová slova
- All-trans retinoic acid, Cyanobacteria, Retinoid-like activity, Retinoids, Teratogenicity, Zebrafish,
- MeSH
- Aphanizomenon patogenita MeSH
- dánio pruhované embryologie genetika MeSH
- embryo nesavčí účinky léků MeSH
- Microcystis patogenita MeSH
- reportérové geny MeSH
- retinoidy biosyntéza toxicita MeSH
- sinice chemie patogenita MeSH
- teratogeny toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- retinoidy MeSH
- teratogeny MeSH
Produced water (PW) represents the largest volume waste stream in oil and gas production operations from most offshore platforms. PW is difficult to monitor as releases are rapidly diluted and concentrations can reach trace levels. The use of passive samplers can over come this. Here polyethylene (PE) was calibrated for a diverse range of PW pollutants. Zebrafish were exposed to dilutions of PW and passive sampler extracts in order to investigate the relationship between freely dissolved chemical concentrations and acute toxic effects. The raw PW had an LC50 of 13% (percentage of PW in the standardized zebrafish medium). Observed non-viable deformations to embryos (at 5 hpf) included heart and yolk edema, head, spine and tail deformations. The dose-response relationship of lethal effects showed that if 0.0041 g of PE is exposed to this PW, then extracted, 50% of exposed D. rerio will suffer lethal effects. The sum of tested freely dissolved concentrations that led to 50% lethal effects (mortality and non-viable deformations) was 2.32 × 10-4 mg/L for PW and 7.92 × 10-2 mg/L for PE. This implies that exposure to raw PW was more toxic than exposure to PE extracts. This toxicity was attributed both to the presence of contaminants as well as PW salinity. Passive samplers are able to detect very low freely dissolved pollutant concentrations which is important for assessing the spatial dilution of PW releases. Bioassays provide complimentary information as they account for all toxic compounds including those that are not taken up by passive samplers.
- Klíčová slova
- Deformations, Monitoring, Polyethylene, Produced water, Zebrafish,
- MeSH
- biotest MeSH
- chemické látky znečišťující vodu analýza chemie MeSH
- monitorování životního prostředí metody MeSH
- odpadní voda chemie MeSH
- průmysl ropy a zemního plynu normy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu MeSH
- odpadní voda MeSH
Herbicides are the most widely used group of pesticides but after reaching water bodies they are able to cause adverse effects on non-target organisms. Different formulations using the same active ingredient are frequently available, which raises the issue of potential influence of different formulation types on herbicide toxicity. The present study evaluated the toxicity and teratogenic effects of the active ingredient clomazone and its two formulations (Rampa® EC and GAT Cenit 36 CS, both containing 360g a.i./l of clomazone) on zebrafish embryos. The crucial difference between the two formulation types is the way of active substance release. This investigation is the first report on zebrafish embryotoxicity of both clomazone and its formulations. The technical active ingredient and formulations caused mortality and diverse teratogenic effects, showing different levels of toxicity. The LC50 values for the technical ingredient, Rampa® EC and GAT Cenit 36 CS were 61.4, 9.6 and 92.5mg a.i./l, respectively. Spontaneous movements in 22 hpf embryos decreased under exposure to both the technical ingredient and formulations. A significant number of underdeveloped embryos was detected after exposure to clomazone and Rampa® EC, while no underdevelopment was noted in embryos exposed to GAT Cenit 36 CS. Exposure to the technical ingredient and formulations led also to a series of morphological changes and interfered with the growth of zebrafish embryos. The EC50 based on detection of edemas, spine and tail tip deformations and gas bladder absence (120hpf) was 12.1, 10.1 and 24.1mg/l for technical clomazone, Rampa® EC and GAT Cenit 36 CS, while teratogenicity index (TI) based on LC50/EC50 ratio was 5.1, 1 and 3.8, respectively. The data in this study showed that the emulsifiable concentrate formulation (Rampa® EC) caused statistically significantly higher toxicity, and the aqueous capsule suspension (GAT Cenit 36 CS) lower toxicity than technical clomazone. It indicates that different formulations with the same active ingredient may have different environmental impacts, which is why risk assessment based only on active ingredient toxicity might not be sufficient in terms of preventing formulation effects on the environment.
- Klíčová slova
- Clomazone, Embryo, Embryotoxicity, Formulations, Zebrafish,
- MeSH
- chemické látky znečišťující vodu toxicita MeSH
- dánio pruhované embryologie MeSH
- embryo nesavčí abnormality účinky léků MeSH
- herbicidy toxicita MeSH
- isoxazoly toxicita MeSH
- oxazolidinony toxicita MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- chemické látky znečišťující vodu MeSH
- clomazone MeSH Prohlížeč
- herbicidy MeSH
- isoxazoly MeSH
- oxazolidinony MeSH
