Fish Embryo Toxicity Research Articles

Impacts of isopyrazam exposure on the development of early-life zebrafish (Danio rerio).

Isopyrazam (IPZ) is a broad spectrum succinate dehydrogenase inhibitor fungicide. Little is known about its potential ecological risks of aquatic organisms recently. The present study examined the embryonic development effects of zebrafish exposed to IPZ under static condition using a fish embryo toxicity test. The lowest observed effect concentration of IPZ was 0.025mg/L in 4-day exposure. Developmental abnormalities, including edema, small head deformity, body deformation and decreased pigmentation, and mortality were observed in zebrafish embryos of 0.05mg/L and higher concentrations, which shown concentration dependency. The heart rate of zebrafish was disrupted by IPZ. Moreover, enzyme and gene experiments shown that IPZ exposure caused oxidative stress of zebrafish. Furthermore, it induced a decrease of succinate dehydrogenase (SDH) enzyme activity and gene transcription level in zebrafish larvae. It can be speculated that IPZ may have a lethal effect on zebrafish, which is accompanied by decreased SDH activity, oxidative stress, and abnormality. These results provide toxicological data about the IPZ on aquatic non-target organisms, which could be useful for further understanding potential environmental risks.

Identification, growth and toxicity assessment of Coolia Meunier (Dinophyceae) from Nova Scotia, Canada

Benthic dinoflagellates of the toxigenic genus Coolia Meunier (Dinophyceae) are known to have a global distribution in both tropical and temperate waters. The type species, C. monotis, has been reported from the Mediterranean Sea, the NE Atlantic and from Rhode Island, USA in the NW Atlantic, whereas other species in the genus have been reported from tropical locations. Coolia cells were observed in algal drift samples collected at seven sites in Nova Scotia, Canada. Clonal isolates were established from four of these locations and identified with light and scanning electron microscopy, then confirmed with genetic sequencing to be C. monotis. This is the first record of this species in Nova Scotia. The isolates were established and incubated at 18 °C under a 14:10 L:D photoperiod with an approximate photon flux density of 50–60 μmol m−2 s−1. Growth experiments using an isolate from Johnston Harbour (CMJH) were carried out at temperatures ranging from 5 to 30 °C under the same photoperiod with an approximate photon flux density of 45–50 μmol m−2 s−1. Cells tolerated temperatures from 5 to 25 °C with optimum growth and mucilage aggregate production between 15 and 20 °C. Methanol extracts of this isolate examined by Liquid Chromatography-Mass Spectrometry (LC–MS) did not show the presence of the previously reported cooliatoxin. Toxic effects were assayed using two zebrafish bioassays, the Fish Embryo Toxicity (FET) assay and the General Behaviour and Toxicity (GBT) assay. The results of this study demonstrate a lack of toxicity in C. monotis from Nova Scotia, as has been reported for other genetically-confirmed isolates of this species. Conditions in which cell growth that could potentially degrade water quality and provide substrate and dispersal mechanisms for other harmful microorganisms via mucilage production are indicated.

Structure‐based design of Nucleosome Binding Peptides for controlling cell function

Proteins that bind the nucleosome, the repetitive unit of chromatin, and the histone H4 tail are critical to establishing chromatin architecture and phenotypic outcomes. A myriad of histone modifications, which strongly induce changes in chromatin structure, have been associated with malignancies and other diseases, highlighting the crucial role of chromatin architectural changes in disease mechanisms. The nucleosome surface was hypothesised as a therapeutic target, however the development, design and function of exogenous nucleosome binding molecules remain unclear.Herein, we performed structure‐based calculations on the nucleosome surface interaction to four peptides (NBMs), which present different nucleosome binding sites, derived from Nucleosome Binding Proteins (NBPs) to analyse the nucleosome sensitivity. Further, we were able to generate a new peptide (GMIP) with two nucleosome binding sites. A series of in vitro studies were performed to verify the impact of all these peptides on the nucleosome, showing differential action on its stability. We also demonstrated that GMIP and a canonical peptide may induce chromatin condensation in vitro. Confocal microscopy using labeled GMIP showed that it penetrates, by a passive mechanism, the cell and binds to chromatin. Cell‐based assays (MTT and FACS) showed that NBMs tested here presented a strong inhibitory activity on tumor cells, but not on non‐tumoral cells, proliferation and viability. It was also shown that these peptides might induce chromatin condensation in vivo. Besides that, the fish embryo toxicity (FET) test showed specificity in actions of NBMs on the ability to induce tissue modifications.Finally, we correlated the structural impact of NBMs to cellular function, providing a unique map on the nucleosome for designing new molecules with important implications. Taking all of this into account, it is clear that the is right to consider the nucleosome surface as a potential therapeutic target to regulate a plethora of cellular disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study of YVO4 as a photocatalyst: Correlation between synthetic route and ecotoxicity

Yttrium orthovanadate (YVO4) powders were synthesized by two different routes: the combustion route (C sample), and the hydrothermal route (H samples). In all samples, oxygen vacancies were observed by Raman, UV–vis diffuse reflectance and photoluminescence (PL) spectroscopy; however they were more significant in C sample. Photocatalytic discoloration of Rhodamine B solutions ranged from 40 to 64% and C and H4 samples presented the highest efficiencies. Although both samples showed similar photocatalytic efficiency, C sample is 11 times more efficient in producing reactive oxygen species than H4 sample, and an interesting effect of the treated solutions was observed in the Fish Embryotoxicity Test, carried out in D. rerio. The C-treated solution was more toxic than the untreated solution, while the H4-treated solution did not show toxicity enhancement. C-treated solution caused a significant mortality of D. rerio embryos beginning at the 4th day of exposure and reached total mortality at the 6th day. Thus, the synthetic route employed in the preparation of C and H4 samples leads to different toxic effects in the treated solutions. These results pointed out that microstructural characteristics and synthetic parameters are not only important for obtaining highly active materials, but they also impact on the toxicity of the effluents.

Microplastic accumulation patterns and transfer of benzo[a]pyrene to adult zebrafish (Danio rerio) gills and zebrafish embryos.

Since only a few studies have investigated effects of microplastics (MPs) by routes other than ingestion, this study was designed to analyze the accumulation patterns and transfer of toxic substances associated with microplastic exposure by simple attachment to (1) adult zebrafish (Danio rerio) gills and (2) zebrafish embryos. Two sizes of fluorescently labelled polymers (1-5 and 10-20 μm) loaded with the model polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) were used to analyze fate, accumulation and transfer of microplastic-associated persistent organic pollutants (POPs) on gills and embryos. Results indicate that microplastics did not permanently accumulate at high amounts in adult zebrafish gills after 6 nor 24 h of incubation: Most particles only superficially adhered to the mucus layer on the filaments, which is constantly being excreted. In contrast, the smaller and heavier MPs (1-5 μm) accumulated in high numbers on the surface of zebrafish egg chorions. In both exposure scenarios, transfer of BaP could be visualized with fluorescence microscopy: A prominent BaP signal was visible both in gill filaments and arches after 6 and 24 h incubation and in zebrafish embryos after exposure to BaP-spiked microplastics. Furthermore, the gill EROD (Ethoxyresorufin-O-deethylase) assay showed a clear trend to CYP 1A (Cytochrom P450 1 A) induction via exposure to BaP-spiked microplastics. However, BaP from spiked microplastics did not reach sufficiently high concentrations to be able to induce morphological effects in the fish embryo toxicity test (FET). In contrast, control exposure to waterborne BaP did induce effects in the FET. As a conclusion, microplastics can also transfer POPs not only via ingestion, but also by simple attachment to epithelia or via the water column. However, further studies are needed to clarify if these interactions are of environmental concern relative to waterborne exposure to toxic substances.

Zebrafish as a Model for Toxicological Perturbation of Yolk and Nutrition in the Early Embryo.

Developmental toxicity assessments often focus on structural outcomes and overlook subtle metabolic differences which occur during the early embryonic period. Deviant embryonic nutrition can result in later-life disease, including diabetes, obesity, and cardiovascular disease. Prior to placenta-mediated nutrient exchange, the human embryo requires maternally supplied nutritional substrates for growth, called yolk. Here, we compare the biology of the human and zebrafish yolk and review examples of toxicant-mediated perturbation of yolk defects, composition, and utilization. Zebrafish embryos, like human embryos, have a protruding yolk sac that serves as a nutritional cache. Aberrant yolk morphology is a common qualitative finding in fish embryotoxicity studies, but quantitative assessment and characterization provides an opportunity to uncover mechanistic targets of toxicant effects on embryonic nutrition. The zebrafish and the study of its yolk sac is an excellent model for uncovering toxicant disruptions to early embryonic nutrition and has potential to discover mechanistic insights into the developmental origins of health and disease.

Differential uptake of and sensitivity to diphenhydramine in embryonic and larval zebrafish

The zebrafish fish embryo toxicity (FET) test is increasingly employed for alternative toxicity studies, yet our previous research identified increased sensitivity of zebrafish slightly older than embryos employed in FET methods (0-4 d postfertilization [dpf]). We identified rapid steady-state accumulation of diphenhydramine across zebrafish embryo and larval stages. However, significantly (p < 0.05) lower accumulation was observed at 48 h compared to 96 h in chorionated and dechorionated embryos (0-4 dpf), but not in zebrafish at 7 to 11 and 14 to 18 dpf. Increased uptake and toxicity of diphenhydramine was further observed in zebrafish at 7 to 11 and 14 to 18 dpf compared with 0-4 dpf embryos with chorion or dechorionated, which indicates that differential zebrafish sensitivity with age is associated with accumulation resulting from gill and other toxicokinetic and toxicodynamic changes during development. Environ Toxicol Chem 2018;37:1175-1181. © 2017 SETAC.

Enhancing the fathead minnow fish embryo toxicity test: Optimizing embryo production and assessing the utility of additional test endpoints

The fathead minnow fish embryo toxicity (FET) test has been identified as a potential alternative to toxicity test methods that utilize older fish. However, several challenges have been identified with the fathead minnow FET test, including: 1) difficulties in obtaining appropriately-staged embryos for FET test initiation, 2) a paucity of data comparing fathead minnow FET test performance to the fathead minnow larval growth and survival (LGS) test and 3) a lack of sublethal endpoints that could be used to estimate chronic toxicity and/or predict adverse effects. These challenges were addressed through three study objectives. The first objective was to optimize embryo production by assessing the effect of breeding group composition (number of males and females) on egg production. Results showed that groups containing one male and four females produced the largest clutches, enhancing the likelihood of procuring sufficient numbers of embryos for FET test initiation. The second study objective was to compare the performance of the FET test to that of the fathead minnow LGS test using three reference toxicants. The FET and LGS tests were similar in their ability to predict the acute toxicity of sodium chloride and ethanol, but the FET test was found to be more sensitive than the LGS test for sodium dodecyl sulfate. The last objective of the study was to evaluate the utility and practicality of several sublethal metrics (i.e., growth, developmental abnormalities and growth- and stress-related gene expression) as FET test endpoints. Developmental abnormalities, including pericardial edema and hatch success, were found to offer the most promise as additional FET test endpoints, given their responsiveness, potential for predicting adverse effects, ease of assessment and low cost of measurement.

Assessment of Ozone or Not-Treated Wastewater Ecotoxicity Using Mechanism-Based and Zebrafish Embryo Bioassays

Toxicity of wastewater treatment plant (WWTP) effluents is usually assessed with standardized bioassays, as e.g. the Fish Embryo Toxicity test (FET), but assessment of sub-lethal toxic effects requires to develop more adapted tests. The present work aimed to add the recording of several sub-lethal endpoints in exposed zebrafish embryo-larval stages in order to increase the sensitivity of residual toxicity evaluation of WWTP effluents using a semi-quantitative method (iFET score). This approach was complemented with the genotoxicity assessment on the exposed embryos, a sub-acute hazard particularly relevant to evaluate a potential chronic toxicity risk of low and multi-contaminated environmental matrices. Additionally, endocrine activities of effluents were quantified using human reporter cell lines. This test battery was applied to the assessment of the residual toxicity of five biological treatment effluents, further treated or not using various ozonation treatments intended to improve pharmaceutical compounds removal. Acute toxicity towards zebrafish embryos was very low. However, iFET score approach proposed was able to reveal residual toxicity through the presence of developmental abnormalities in all samples tested. Additionally, a low residual genotoxicity was measured in embryos exposed to two of the WWTP effluents when all excepted one exhibited a residual endocrine activity potential in the ng/L range providing complementary information on the occurrence of endocrine active chemicals and their reduction by different processes. To sum up, such a simplified and ecologically relevant test battery was found sensitive enough to characterize and differentiate various residual effluent’s ecotoxicity at contaminant levels of environmental concern.

Electrochemical simulation of triclosan metabolism and toxicological evaluation

Tricolsan (TCS), an antimicrobial agent, is considered as emerging pollutant due to its wide dispersive use in personal care products and high aquatic toxicity. In the present study, phase I metabolism of triclosan was investigated through laboratory electrochemical simulation studies. The products formed in the electrochemical (EC) cell were identified by online and offline coupling with QTRAP and high-resolution FTICR mass spectrometers, respectively. The sequential formation and disappearance of each product, with the continuous increase of voltage from 0 to 3500 mV, was observed to reveal the transformation pathways of TCS. The toxic potential of TCS and the identified products was estimated using Quantitative structure–activity relationship (QSAR) modeling on 16 target proteins. The toxicity change of TCS during simulated metabolism and toxicological effects of reaction mixture were assessed by Fish embryo toxicity (FET) test (Danio rerio) and quantitative real-time polymerase chain reaction (qPCR). Eight metabolites formed during the simulated metabolism of TCS mainly via the mechanisms of hydroxylation, ether-bond cleavage and cyclization. In FET test, the reaction mixture (LC50, 48h=1.28 mg/L) after electrochemical reactions showed high acute toxicity on zebrafish embryos, which was comparable to that of triclosan (LC50, 48h=1.34 mg/L). According to the modeling data, less toxic products formed only via ether-bond cleavage of TCS while the products formed through other mechanisms showed high toxicity. AhR-mediated dioxin-like effects on zebrafish embryos, such as developmental retardation in skeleyton and malformations in cardiovascular system, were also observed after exposure to the TCS reaction mixture in FET test. Activation of the AhR by the reaction mixture in zebrafish embryos was further proved in cyp1a gene expression analysis.

Towards more ecological relevance in sediment toxicity testing with fish: Evaluation of multiple bioassays with embryos of the benthic weatherfish (Misgurnus fossilis)

The effects of sediment contamination on fish are of high significance for the protection of ecosystems, human health and economy. However, standardized sediment bioassays with benthic fish species, that mimic bioavailability of potentially toxic compounds and comply with the requirements of alternative test methods, are still scarce. In order to address this issue, embryos of the benthic European weatherfish (Misgurnus fossilis) were exposed to freeze-dried sediment (via sediment contact assays (SCA)) and sediment extracts (via acute fish embryo toxicity tests) varying in contamination level. The extracts were gained by accelerated solvent extraction with (i) acetone and (ii) pressurized hot water (PHWE) and subsequently analyzed for polycyclic aromatic hydrocarbons, polychlorinated biphenyls and polychlorinated dibenzodioxins and dibenzofurans. Furthermore, embryos of the predominately used zebrafish (Danio rerio) were exposed to extracts from the two most contaminated sediments. Results indicated sufficient robustness of weatherfish embryos towards varying test conditions and sensitivity towards relevant sediment-bound compounds. Furthermore, a compliance of effect concentrations derived from weatherfish embryos exposed to sediment extracts (96h-LC50) with both measured gradient of sediment contamination and previously published results was observed. In comparison to zebrafish, weatherfish embryos showed higher sensitivity to the bioavailability-mimicking extracts from PHWE but lower sensitivity to extracts gained with acetone. SCAs conducted with weatherfish embryos revealed practical difficulties that prevented an implementation with three of four sediments tested. In summary, an application of weatherfish embryos, using bioassays with sediment extracts from PHWE might increase the ecological relevance of sediment toxicity testing: it allows investigations using benthic and temperate fish species considering both bioavailable contaminants and animal welfare concerns.

In search of a comprehensible set of endpoints for the routine monitoring of neurotoxicity in vertebrates: sensory perception and nerve transmission in zebrafish (Danio rerio) embryos.

In order to develop a test battery based on a variety of neurological systems in fish, three sensory systems (vision, olfaction, and lateral line) as well as nerve transmission (acetylcholine esterase) were analyzed in zebrafish (Danio rerio) embryos with respect to their suitability as a model for the screening of neurotoxic trace substances in aquatic ecosystems. As a selection of known or putative neurotoxic compounds, amidotrizoic acid, caffeine, cypermethrin, dichlorvos, 2,4-dinitrotoluene, 2,4-dichlorophenol, 4-nonylphenol, perfluorooctanoic acid, and perfluorooctane sulfonic acid were tested in the fish embryo test (OECD test guideline 236) to determine EC10 values, which were then used as maximum test concentration in subsequent neurotoxicity tests. Whereas inhibition of acetylcholinesterase was investigated biochemically both in vivo and in vitro (ex vivo), the sensory organs were studied in vivo by means of fluorescence microscopy and histopathology in 72- or 96-h-old zebrafish embryos, which are not regarded as protected developmental stages in Europe and thus - at least de jure- represent alternative test methods. Various steps of optimization allowed this neurotoxicity battery to identify neurotoxic potentials for five out of the nine compounds: Cypermethrin and dichlorvos could be shown to specifically modulate acetylcholinesterase activity; dichlorvos, 2,4-dichlorophenol, 4-nonylphenol, and perfluorooctane sulfonic acid led to a degeneration of neuromasts, whereas both vision and olfaction proved quite resistant to concentrations ≤ EC10 of all of the model neurotoxicants tested. Comparison of neurotoxic effects on acetylcholinesterase activity following in vivo and in vitro (ex vivo) exposure to cypermethrin provided hints to a specific enzyme-modulating activity of pyrethroid compounds. Enhancement of the neuromast assay by applying a simultaneous double-staining procedure and implementing a 4-scale scoring system (Stengel et al. 2017) led to reduced variability of results and better statistical resolution and allowed to differentiate location-dependent effects in single neuromasts. Since acetylcholinesterase inhibition and neuromast degeneration can be analyzed in 72- and 96-h-old zebrafish embryos exposed to neurotoxicants according to the standard protocol of the fish embryo toxicity test (OECD TG 236), the fish embryo toxicity test can be enhanced to serve as a sensitive neurotoxicity screening test in non-protected stages of vertebrates.

Toxicological profile and acetylcholinesterase inhibitory potential of Palicourea deflexa, a source of β-carboline alkaloids

Palicourea genus is chemically and taxonomically close to Psychotria genus, a well-known source of neuroactive alkaloids. It has been previously reported the pharmacological potential of these alkaloids in some targets related to the neurodegenerative process. In this context, this study was carried out in order to evaluate the toxic effects and acetylcholinesterase (AChE) inhibitory potential of Palicourea deflexa fraction of total alkaloids (FTA). P. deflexa FTA was analyzed by means of HPLC-DAD and HRMS-ESI. We performed toxicological screening through Fish Embryo Toxicity (FET) test using zebrafish embryo and abnormal developmental phenotypes were recorded daily. For AChE inhibition, zebrafish brains were used as enzymatic source and formation of thiolate dianion of 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was used to monitor acetylthiocholine hydrolysis. Lineaweaver–Burk double reciprocal plots were used to indicate mode of inhibition. Chemical analysis of the P. deflexa FTA allowed the identification of the main compound as harman-3-carboxylic acid. This fraction was evaluated in vivo for its toxicological effect. The zebrafish embryo test indicated that the FTA has a lethal concentration of 50% (LC50)=72.18μg/mL. Further, the FTA was evaluated for its AChE inhibitory profile, demonstrating an inhibitory concentration of 50% (IC50) of 50.65μg/mL. Lineaweaver–Burk double reciprocal plots indicated a mixed mode of inhibition. It is reported for the first time the toxicological and pharmacological profile of the alkaloid fraction of Palicourea deflexa in zebrafish models.

Effect-based assessment of toxicity removal during wastewater treatment

Wastewaters contain complex mixtures of chemicals, which can cause adverse toxic effects in the receiving environment. In the present study, the toxicity removal during wastewater treatment at seven municipal wastewater treatment plants (WWTPs) was investigated using an effect-based approach. A battery of eight bioassays was applied comprising of cytotoxicity, genotoxicity, endocrine disruption and fish embryo toxicity assays. Human cell-based CALUX assays, transgenic larval models and the fish embryo toxicity test were particularly sensitive to WWTP effluents. The results indicate that most effects were significantly reduced or completely removed during wastewater treatment (76–100%), while embryo toxicity, estrogenic activity and thyroid disruption were still detectable in the effluents suggesting that some harmful substances remain after treatment. The responsiveness of the bioassays was compared and the human cell-based CALUX assays showed highest responsiveness in the samples. Additionally, the fish embryo toxicity test and the transgenic larval models for endocrine disrupting effects showed high responsiveness at low sample concentrations in nearly all of the effluent samples. The results showed a similar effect pattern among all WWTPs investigated, indicating that the wastewater composition could be rather similar at different locations. There were no considerable differences in the toxicity removal efficiencies of the treatment plants and no correlation was observed with WWTP characteristics, such as process configuration or sludge age. This study demonstrated that a biotest battery comprising of multiple endpoints can serve as a powerful tool when assessing water quality or water treatment efficiency in a holistic manner. Rather than analyzing the concentrations of a few selected chemicals, bioassays can be used to complement traditional methods of monitoring in the future by assessing sum-parameter based effects, such as mixture effects, and tackling chemicals that are present at concentrations below chemical analytical detection limits.

In vitro genotoxicity and in vivo subchronic evaluation of the anti-inflammatory pyrazole compound LQFM021

Scientific evidences have highlighted 5-(1-(3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM021) as a promising anti-inflammatory, analgesic and antinociceptive agent due to its effects on peripheral opioid receptors associated with activation of the nitric oxide/cGMP/KATP pathway. Despite these important pharmacological findings, toxicity data of LQFM021 are scarce. Thus, this study investigated the in vitro genotoxicity of LQFM021 through cytokinesis-block micronucleus assay (OECD Nº 487/2014). Moreover, zebrafish model was used to assess the embryotoxicity potential of LQFM021 using fish embryo toxicity test (OECD Nº 236/2013) with extended exposure to evaluate subchronic larval development. In vivo subchronic toxicity of LQFM021 in rats (OECD Nº 407/2008) was also conducted. This compound at the lower concentrations tested (3.1 and 31 μg/mL) did not promote changes in micronuclei frequency in HepG2 cells. However, in the higher concentrations of LQFM021 (310 and 620 μg/mL) triggered a significant increase of micronucleated HepG2 cells, showing an alert signal of potential genotoxicity. Regarding the oral treatment of rats with LQFM021 (62.5, 125 or 250 mg/kg) for 28 days, the main findings showed that LQFM021 promoted renal and liver changes in a dose-dependent manner, being irreversible damage for kidneys while liver tissue showed a recovery after 14 days post treatment. Regarding embryotoxicity, although the lower concentrations used did not show toxicity, the concentration of LQFM021 (39.8 and 100 mg/L) promoted malformations in zebrafish embryo-larvae stage, in especial cardiac tissue changes. In conclusion, anti-inflammatory compound LQFM021 seems to have some limiting factors as a new therapeutic option to be used orally and in high repeated doses, related to those found in the non-steroidal anti-inflammatory drugs (NSAIDs).

Investigation of a Brazilian tannery effluent by means of zebra fish (Danio rerio) embryo acute toxicity (FET) test

ABSTRACTTannery effluents consist of a complex chemical composition not only limited to primary pollutants, which also require biological detection as these compounds may produce adverse effects. The fish embryo toxicity (FET) test with Danio rerio is an alternative method in hazard and risk assessment for determination of chemical-mediated effects. The aim of this investigation was to use the FET test to detect compounds and consequent effects in Brazilian tannery effluents. Samples were collected from the inlet and outlet of the effluent treatment plant at a tannery located in Restinga, São Paulo, Brazil. The toxicological effects were assessed using FET assay for a period of 144 hr using indices such as (1) coagulation of fertilized eggs, (2) lack of detachment of tail-bud from yolk sac, (3) absence of spontaneous movement, (4) yolk sack edema, (5) malformation of the tail, (6) scoliosis, and (7) deformation of swim bladder in the embryos. Data showed that effluent treatment plant exposure produced acute toxicity in D. rerio embryos as evidenced by coagulation of fertilized eggs in up to 5% of all diluted samples 24 hr post fertilization for inlet effluent samples compared to 100% coagulation for outlet samples. Results demonstrated that these effects may not be attributed to metals, but to other non-detected components, such as dyes, pigments, biocides, carriers, surfactants, or other organic compounds that might be present in these complex mixtures. The use of D. rerio embryos was found to be useful as an additional tool for ecotoxicity testing to assess the potential environmental acute toxicity influence of tannery effluents.

Evaluation of water quality during successive severe drought years within Microcystis blooms using fish embryo toxicity tests for the San Francisco Estuary, California

In the San Francisco Estuary, California, the largest estuary on the Pacific Coast of North America, the frequency and intensity of drought and associated cyanobacteria blooms are predicted to increase with climate change. To assess the impact of water quality conditions on estuarine fish health during successive severe drought years with Microcystis blooms, we performed fish embryo toxicity testing with Delta Smelt and Medaka. Fish embryos were exposed to filtered ambient water collected from the San Francisco Estuary during the Microcystis bloom season in 2014 and 2015, the third and fourth most severe recorded drought years in California. Medaka embryos incubated in filtered ambient waters exhibited high mortality rates (>77%), which was mainly due to bacterial growth. Medaka mortality data was negatively correlated with chloride, and positively correlated with water temperature, total and dissolved organic carbon, and ambient and net chlorophyll a concentration. Delta Smelt embryo mortality rates were lower (<42%) and no prominent seasonal or geographic trend was observed. There was no significant correlation between the Delta Smelt mortality data and water quality parameters. Aeromonas was the dominant bacteria that adversely affected Medaka. The growth of Aeromonas was suppressed when salinity was greater than or equal to 1psu and resulted in a significant reduction in mortality rate. Bacterial growth test demonstrated that the lysate of Microcystis cells enhanced the growth of Aeromonas. Toxin production by Microcystis is a major environmental concern, however, we conclude that dissolved substances released from Microcystis blooms could result in water quality deterioration by promoting growth of bacteria. Furthermore, a distinctive developmental deformity was observed in Medaka during the toxicity tests; somite formation was inhibited at the same time that cardiogenesis occurred and the functional heart was observed to be beating. The exact cause of the embryonic developmental deformity is still unknown.

Effects of copper oxide nanoparticles and copper ions to zebrafish (Danio rerio) cells, embryos and fry

The use of engineered metal nanoparticles (NPs) is continuously increasing and so is the need for information regarding their toxicity. This study compares the toxicity of CuO NPs with ionic Cu in three zebrafish model systems; zebrafish hepatoma cell line (ZFL), fish embryo toxicity test (FET) and fry locomotion. In the ZFL tests, no significant cytotoxicity (cell death, decreased metabolic or cell membrane integrity) was detected for either treatment, though both significantly affected reactive oxygen species (ROS) production. Embryo mortality was affected by both Cu ions and CuO NPs with similar concentration-response relationships, whereas only Cu ions affected fry mortality (24h LC50≈30μM, ≈2mgCuL−1 for Cu ions and no significant mortality observed at up to 200μM, 12.7mgCuL−1 for CuO NP). Both Cu forms increased fry swimming activity during light cycles and decreased activity during dark cycles: Cu ions had significant impact at lower concentrations than CuO NPs. The implications are that Cu ions generally are more toxic than CuO NPs to embryos and fry but there is a marked difference in toxicity among the different zebrafish model systems. Metal NPs release into the environment may have adverse effects on fish and other aquatic organisms.

Assessing the ecotoxicity of potentially neurotoxic substances – Evaluation of a behavioural parameter in the embryogenesis of Danio rerio

The pollution of the aquatic environment is currently characterised by a large number of contaminants, especially by mixtures of micro-pollutants including neurotoxins. The ecotoxicological consequences of this burden are not yet assessable. Within the present study, a new test method was applied which evaluates behavioural changes in zebrafish (Danio rerio) embryos to quantify the neurotoxic effect of selected chemicals. Changes in the frequency of spontaneous tail movements – a parameter of locomotion – occurring during embryogenesis was assessed as parameter for neurotoxic effects. Embryos were exposed in 24-microwell plates to neurotoxic compounds. Behaviour was examined after 24 h of exposure by videotaping and quantifying spontaneous locomotion. Additionally, acute toxicity was determined after 48 h of exposure by utilising the fish embryo toxicity test. Abamectin, emamectin benzoate, chlorpyrifos-oxon and carbamazepine were analysed using both: the new and classic acute test methods. The results showed the neurotoxic effect of the substances. Furthermore, an increased sensitivity compared to acute toxicity data was shown. The aim of the present study, to illustrate the sensitivity and specificity of the established endpoint to reliably assess adverse, neurotoxic effects of compounds on the model organism Danio rerio and to apply a new test method was achieved. Therefore, the present study is a substantial contribution to an effect-based risk assessment of contaminants in aquatic ecosystems.

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.