Background
Zebrafish have been increasingly used for monitoring and assessing the effects of different contaminants in the aquatic environments. In the present study, zebrafish embryos and larvae ...were used to study the effects of the insecticide diazinon and the herbicide diuron in regard to occurrence of oxidative stress-related cellular responses, multixenobiotic resistance (MXR)-related efflux transporter activity—which represents the first line of defense against xenobiotics in many aquatic organisms—and responses of different molecular and biochemical biomarkers.
Results
The recently established non-invasive quantitative plate assay, which uses the fluorescent probes CM-H
2
DCFDA and CellTracker™ Green CMFDA and measures the fluorescence in whole zebrafish larvae, was applied to assess changes in reactive oxidative species (ROS) and glutathione (GSH) after exposure to the investigated pesticides. The results showed a significant increase of GSH after 1 h exposure of zebrafish larvae to both diazinon and diuron. Regarding the ROS induction, no significant increases in fluorescence could be detected after 2 h exposure to the investigated pesticides. Applying a newly adapted assay for MXR activity, it was determined that diuron caused no change after a 24-h exposure, but caused a significant induction of MXR activity after a 48-h exposure (indicated by a decreased amount of accumulated rhodamine B). On the other hand, diazinon caused an inhibition of MXR activity after both 24 h and 48 h exposure (indicated by an increased amount of accumulated rhodamine B). Regarding the biomarkers, different set-ups and exposure periods were applied and both molecular (gene expression) and biochemical (enzymatic activities) responses were assessed. Diazinon caused an inhibition of carboxylesterase (CES) and acetylcholinesterase (AChE) activity in zebrafish larvae, diuron inhibited AChE activity in in vitro testing, and both pesticides significantly affected gene expression and activities of some of the cytochrome P450 (CYP) family enzymes.
Conclusions
The obtained results show various effects of the investigated pesticides and will help to elucidate how aquatic animals cope with pesticides present in their environment. Additionally, the recently developed fluorescence-based assay and the newly adapted MXR activity assay proved to be useful tools for ecotoxicological risk assessment to further investigate pesticide effects.
Background
Floodplains are biodiversity hotspots and provide numerous ecosystem services. In recent decades, however, 70–90% of Europe’s floodplains have been structurally degraded. Accordingly, many ...(inter-)national programs aim to restore and protect floodplain ecosystems. The success of such measures also depends on the chemical contamination, especially of floodplain soils and sediments, which serve as sinks and sources for a variety of pollutants. In this study, we assess the current ecotoxicological status of a floodplain restoration site along the Main River (Frankfurt am Main, Germany) and estimate its development potential with respect to the influence of a local industrial plant and potential legacy contaminations. We therefore use in vitro effect-based methods (EBMs) testing for baseline toxicity, mutagenicity, dioxin-like and estrogenic activities, coupled with chemical analysis.
Results
Of all water bodies analyzed, the overall toxicity was highest in two flood depressions. In the respective water phase, estrogenic activities exceeded the environmental quality standard and sediment samples were positive for all tested endpoints. Chemical analysis of these sediments revealed high concentrations of polycyclic aromatic hydrocarbons. Soil samples from frequently flooded areas showed the highest mutagenic potential for both frameshift and point mutations with and without metabolic activation. The industrial effluent showed baseline toxic, mutagenic, and dioxin-like activities, that were highly diluted in the Main River. In turn, most of the sediment samples downstream of the industrial discharge showed significantly elevated baseline toxic, estrogenic and dioxin-like activities as well as increased chemical contamination.
Conclusion
Based on the results of this study, we rate the overall ecotoxicological status of a recently established tributary and groundwater-fed ponds as good, and identified two flood depressions near the Main River as hot spots of contamination. We assume that the observed mutagenicity in the floodplain soils is related to legacy contaminations from former aniline and azo dye production. In terms of the development potential of the floodplain restoration site, we emphasize considering the remobilization of pollutants from these soils and suppose that, in the long term, pollution of the Main River and the local industrial plant may negatively impact sediment quality in its tributaries. With this study, we confirmed the utility of in vitro EBMs for identifying chemically and ecotoxicologically relevant sites.
Chemical pollution resulting from pesticide usage has been a continuous issue since the 1960s, despite comprehensive European Union legislation designed to safeguard human health and the environment ...from the adverse effects of pesticides. While regulatory risk assessments primarily focus on the active ingredients, recent research indicates ecotoxicological impacts of commercial preparations on non-target organisms, particularly within the soil ecosystem where key species such as earthworms play a vital role in maintaining soil quality and fertility. Therefore, the aim of this study was the assessment of the long-term effects of the following respective commercial preparations: the insecticides Sumialfa (esfenvalerate) and Calypso (thiacloprid), as well as the herbicides Frontier (dimethenamid-
p
) and Filon (prosulfocarb) on the earthworm
Eisenia andrei
in standardized soil during long-term exposures of 7, 14, and 28 days. To study the possible effects on different levels of biological organization, enzymatic biomarkers: acetylcholinesterase (AChE), carboxylesterase (CES) glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx); non-enzymatic biomarkers: multixenobiotic resistance activity (MXR), levels of glutathione (GSH), and reactive oxygen species (ROS) as well as reproductive success were investigated. While Calypso appeared to be the least toxic substance, all pesticides showed significant effect on multiobiomarker response in
E. fetida
. That being said, the response of MXR activity was significantly altered by all tested pesticides indicating MXR being the most sensitive endpoint of the present research. Recovery of MXR was observed after 28 days, however, only in case of exposure to Filon, while the recovery of CAT activity was recorded after 28 days as well, subsequent to Sumialfa exposure. Reproductive success was negatively impacted regarding the Frontier and Sumialfa exposure at the highest concentration (100 mg/kg) reflected in reduced number of cocoons, while only the exposure to Frontier (100 mg/kg) reduced the number of juveniles. Based on the results, it is important to include commercial pesticide formulations in pesticide risk assessments. The toxicity classifications of the studied pesticides suggest the potential detrimental consequences to the key soil species in terrestrial ecosystems at various concentrations. Future studies should include other soil species as well as investigation of higher levels of biological organization, i.e., behavioral endpoints, to determine the potential risks to terrestrial ecosystems.
Background
The fish embryo acute toxicity (FET) test with the zebrafish (
Danio rerio
) was developed to assess the acute fish toxicity of chemicals or environmental samples as a replacement for the ...Acute Fish Test (AFT) with juvenile fish. However, the FET is not yet established in the regulatory context. One reason is the (postulated) difference between the biotransformation capacities of embryos and juvenile fish.
The present study was designed to develop a procedure for external metabolization of test substances prior to testing in the FET. The workflow allows simultaneous exposure of the embryos to the maternal substances and their potential metabolites throughout the entire exposure period. After a 2 h incubation of the samples at 37 °C with non-toxic concentrations of a rat liver S9 homogenate or an animal-free (ewoS9R) metabolization system, freshly fertilized zebrafish embryos are added and incubated up to 120 h post-fertilization at 26 °C. Five biotransformable model substances (allyl alcohol, benzoapyrene (BaP), chlorpyrifos (CP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and bisphenol A (BPA)) were evaluated for embryotoxicity with and without external metabolization.
Results
Only for allyl alcohol, external metabolization with both rat S9 and ewoS9R resulted in significantly higher embryotoxicity than under non-premetabolized conditions and, thus, in a better correlation of FET and AFT data. For BaP, CP, TDCPP and BPA, there was no relevant difference between data derived from the FET (with and without pre-metabolization) and literature AFT data; even though the FET results with and without pre-metabolization differed significantly for BPA (with rat S9 and ewoS9R) and TDCPP (rat S9 only).
Conclusions
External pre-metabolization appears a promising add-on to the FET protocol to improve the correlation with AFT data of certain biotransformable substances and might help to strengthen the FET as an alternative to the AFT and finally to reduce or replace sentient animals used for acute fish toxicity data in the regulatory context.
Graphical Abstract
This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The ...assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter range. A researcher familiar with the procedure can process up to 160 samples with four wells each within 3 d. The series described uses four plates with three concentrations per sample, which can be easily scaled to accommodate different sample sizes.
Green Toxicology refers to the application of predictive toxicology in the sustainable development and production of new less harmful materials and chemicals, subsequently reducing waste and ...exposure. Built upon the foundation of “Green Chemistry” and “Green Engineering”, “Green Toxicology” aims to shape future manufacturing processes and safe synthesis of chemicals in terms of environmental and human health impacts. Being an integral part of Green Chemistry, the principles of Green Toxicology amplify the role of health-related aspects for the benefit of consumers and the environment, in addition to being economical for manufacturing companies. Due to the costly development and preparation of new materials and chemicals for market entry, it is no longer practical to ignore the safety and environmental status of new products during product development stages. However, this is only possible if toxicologists and chemists work together early on in the development of materials and chemicals to utilize safe design strategies and innovative in vitro and in silico tools. This paper discusses some of the most relevant aspects, advances and limitations of the emergence of Green Toxicology from the perspective of different industry and research groups. The integration of new testing methods and strategies in product development, testing and regulation stages are presented with examples of the application of in silico, omics and in vitro methods. Other tools for Green Toxicology, including the reduction of animal testing, alternative test methods, and read-across approaches are also discussed.
Pesticides contribute to this reduction of biodiversity in ecosystems. Obviously, environmental risk assessment did not prevent adverse pesticide effects on non-target organisms. This called for an ...identification of processes that are relevant to extrapolate from simplified investigations to the reality of pesticide effects in the field, one of the prominent research areas at the SETAC GLB since two decades. We identify research areas that are relevant to link toxicant effects from test systems with the ecosystem to increase the realism of pesticide risk assessment.
Endpoints of planar halogenated aromatic hydrocarbon (pHAH) and polycyclic aromatic hydrocarbon (PAH) toxicity are mediated
via activation of the aryl hydrocarbon receptor (AhR) followed by ...activation of the so called “AhR-battery” of genes including the cytochrome P450 1 (CYP1) isoforms. The aim of this study was to develop a method to identify CYP1 activity in early life-stages of zebrafish (
Danio rerio) in order to elucidate the spatio-temporal pattern of basal and induced CYP1 activities. Preliminary experiments with the fish embryo toxicity test (FET) were carried out to determine toxic effect thresholds of the AhR agonist β-naphthoflavone. To assess basal and β-naphthoflavone-induced CYP1 activity during early life-stages of zebrafish, the commonly used 7-ethoxyresorufin-
O-deethylase (EROD) assay was developed further for use in confocal laser scanning microscopy (CLSM) and spectrometry. Following exposure to selected cytochrome P450 inducers, zebrafish embryos were dechorionated, anaesthetized and inspected
in vivo under the CLSM. Alternatively, embryos were homogenized, and EROD activity was measured using classical spectrometry
in vitro. CLSM of CYP-induced fluorescence allowed for the
in vivo detection of CYP1 enzyme activity down to the cellular level as early as in the gastrulation stage. Basal and induced CYP1 activity was detected at all time points examined from 8
h post-fertilization to early adulthood and showed a highly dynamic spatio-temporal pattern throughout zebrafish development. Basal and induced EROD activity was prominent in tissues of the cardiovascular system, the urinary tract, the digestive system, and parts of the brain as well as in the central portion of the eye and the otic vesicle during distinct stages of development. The differentiation between constitutive and induced spatio-temporal patterns of CYP1 activity even as early as the gastrula stage provide further insights into the endogenous role of CYP1 activity.
In situ burning (ISB) is discussed to be one of the most suitable response strategies to combat oil spills in extreme conditions. After burning, a highly viscous and sticky residue is left and may ...over time pose a risk of exposing aquatic biota to toxic oil compounds. Scientific information about the impact of burn residues on the environment is scarce. In this context, a comprehensive ISB field experiment with approx. 1000L IFO 180 was conducted in a fjord in Greenland. The present study investigated the toxicity of collected ISB residues to early life stages of zebrafish (
Danio rerio
) as a model for potentially exposed pelagic organisms. The toxicity of ISB residues on zebrafish embryos was compared with the toxicity of the initial (unweathered) IFO 180 and chemically dispersed IFO 180. Morphological malformations, hatching success, swimming behavior, and biomarkers for exposure (CYP1A activity, AChE inhibition) were evaluated in order to cover the toxic response on different biological organization levels. Across all endpoints, ISB residues did not induce greater toxicity in zebrafish embryos compared with the initial oil. The application of a chemical dispersant increased the acute toxicity most likely due to a higher bioavailability of dissolved and particulate oil components. The results provide insight into the adverse effects of ISB residues on sensitive life stages of fish in comparison with chemical dispersant application.
The harpacticoid copepod Nitocra spinipes has become a popular model species for toxicity testing over the past few decades. However, the combined influence of temperature and food shortage, two ...climate change-related stressors, has never been assessed in this species. Consequently, effects of three temperatures (15, 20 and 25°C) and six food regimes (between 0 and 5 × 105 algal cells/mL) on the life cycle of N. spinipes were examined in this study. Similarly to other copepod species, development times and brood sizes decreased with rising temperatures. Mortality was lowest in the 20°C temperature setup, indicating a close-by temperature optimum for this species. Decreasing food concentrations led to increased development times, higher mortality and a reduction in brood size. A sex ratio shift toward more females per male was observed for increasing temperatures, while no significant relationship with food concentration was found. Temperature and food functions for each endpoint were integrated into an existing individual-based population model for N. spinipes which in the future may serve as an extrapolation tool in environmental risk assessment. The model was able to accurately reproduce the experimental data in subsequent verification simulations. We suggest that temperature, food shortage, and potentially other climate change-related stressors should be considered in environmental risk assessment of chemicals to account for non-optimal exposure conditions that may occur in the field. Furthermore, we advocate combining in vivo bioassays with population modeling as a cost effective higher tier approach to assess such considerations.
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