RESUMO
Effect-directed analysis (EDA) is a powerful strategy to identify biologically active compounds in environmental samples. However, in current EDA studies, fractionation and handling procedures are laborious, consist of multiple evaporation steps, and thus bear the risk of contamination and decreased recoveries of the target compounds. The low resulting throughput has been one of the major bottlenecks of EDA. Here, we propose a high-throughput EDA (HT-EDA) work-flow combining reversed phase high-performance liquid chromatography fractionation of samples into 96-well microplates, followed by toxicity assessment in the micro-EROD bioassay with the wild-type rat hepatoma H4IIE cells, and chemical analysis of bioactive fractions. The approach was evaluated using single substances, binary mixtures, and extracts of sediment samples collected at the Three Gorges Reservoir, Yangtze River, China, as well as the rivers Rhine and Elbe, Germany. Selected bioactive fractions were analyzed by highly sensitive gas chromatography-atmospheric pressure laser ionization-time-of-flight-mass spectrometry. In addition, we optimized the work-flow by seeding previously adapted suspension-cultured H4IIE cells directly into the microplate used for fractionation, which makes any transfers of fractionated samples unnecessary. The proposed HT-EDA work-flow simplifies the procedure for wider application in ecotoxicology and environmental routine programs.
Assuntos
Dioxinas , Sedimentos Geológicos/química , Animais , Bioensaio , Ecotoxicologia , Rios/química , Poluentes Químicos da ÁguaRESUMO
The potential to bioconcentrate is generally considered to be an unwanted property of a substance. Consequently, chemical legislation, including the European REACH regulations, requires the chemical industry to provide bioconcentration data for chemicals that are produced or imported at volumes exceeding 100 tons per annum or if there is a concern that a substance is persistent, bioaccumulative, and toxic. For the filling of the existing data gap for chemicals produced or imported at levels that are below this stipulated volume, without the need for additional animal experiments, physiologically-based toxicokinetic (PBTK) models can be used to predict whole-body and tissue concentrations of neutral organic chemicals in fish. PBTK models have been developed for many different fish species with promising results. In this study, we developed PBTK models for zebrafish (Danio rerio) and roach (Rutilus rutilus) and combined them with existing models for rainbow trout (Onchorhynchus mykiss), lake trout (Salvelinus namaycush), and fathead minnow (Pimephales promelas). The resulting multispecies model framework allows for cross-species extrapolation of the bioaccumulative potential of neutral organic compounds. Predictions were compared with experimental data and were accurate for most substances. Our model can be used for probabilistic risk assessment of chemical bioaccumulation, with particular emphasis on cross-species evaluations.
Assuntos
Peixes , Modelos Biológicos , Toxicocinética , Poluentes Químicos da Água/farmacocinética , Animais , Compostos Orgânicos , Poluentes Químicos da Água/química , Poluentes Químicos da Água/toxicidadeRESUMO
The construction of the Three Gorges Dam (TGD) in the Yangtze River raises great concern in ecotoxicological research since large amounts of pollutants enter the Three Gorges Reservoir (TGR) water bodies after TGD impoundment. In this work, effect-directed analysis (EDA), combining effect assessment, fractionation procedure, and target and nontarget analyses, was used to characterize aryl hydrocarbon receptor (AhR) agonists in sediments of the TGR. Priority polycyclic aromatic hydrocarbons (PAHs) containing four to five aromatic rings were found to contribute significantly to the overall observed effects in the area of Chongqing. The relatively high potency fractions in the Kaixian area were characterized by PAHs and methylated derivatives thereof and heterocyclic polycyclic aromatic compounds (PACs) such as dinaphthofurans. Benzothiazole and derivatives were identified as possible AhR agonists in the Kaixian area based on nontarget liquid chromatography-high resolution mass spectrometry (LC-HRMS). To our knowledge, this study is the first one applying the EDA approach and identifying potential AhR agonists in TGR.
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The European REACH regulation requires the use of animal experimentation to assess the risk of industrial chemicals. However, the 3R principle (reduction, replacement, refinement) demands the use of suitable alternative test methods. Many dossiers submitted for the authorization of chemicals have attempted to provide the required data without performing new experiments, relying heavily on in silico methods; in vitro assays were scarcely used. We propose a methodology that uses physiologically based toxicokinetic (PBTK) models to extrapolate in vitro data to the in vivo level. We collected experimental results for in vitro and in vivo ethoxyresorufin-O-deethylase and vitellogenin induction following chemical exposure and compared those results with model predictions. We found that the predictive power of aqueous chemical concentrations was limited; median effect concentrations (EC50s) based on internal concentrations in fish correlated better with in vitro EC50s. Our data show that in vitro assays could offer a substitute for fish studies when combined with PBTK models.
Assuntos
Modelos Biológicos , Oncorhynchus mykiss/metabolismo , Poluentes Químicos da Água/farmacocinética , Animais , Simulação por Computador , Citocromo P-450 CYP1A1/biossíntese , Indução Enzimática/efeitos dos fármacos , Toxicocinética , Vitelogeninas/biossíntese , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
The implementation of in vitro bioassays for the screening of dioxin-like compounds (DLCs) into management guidelines of dredged material is of increasing interest to regulators and risk assessors. This study reports on an intra- and inter-laboratory comparison study between four independent laboratories. A bioassay battery consisting of RTL-W1 (7-ethoxy-resorufin-O-deethylase; EROD), H4IIE (micro-EROD), and H4IIE-luc cells was used to assess aryl hydrocarbon receptor-mediated effects of sediments from two major European rivers, differently contaminated with DLCs. Each assay was validated by characterization of its limit of detection (LOD) and quantification (LOQ), z-factor, reproducibility, and repeatability. DLC concentrations were measured using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) and compared to bioassay-specific responses via toxicity equivalents (TEQs) on intra- and inter-laboratory levels. The micro-EROD assay exhibited the best overall performance among the bioassays. It was ranked excellent (z-factor = 0.54), reached a repeatability > 75%, was highly comparable (r 2 = 0.87) and reproducible (83%) between two laboratories, and was well correlated (r 2 = 0.803) with TEQs. Its LOD and LOQ of 0.5 and 0.7 pM 2,3,7,8-TCDD, respectively, approached LOQs of HRGC/HRMS measurements. In contrast, cell lines RTL-W1 and H4IIE-luc produced LODs > 0.7 pM 2,3,7,8-TCDD, LOQs > 1.7 pM 2,3,7,8-TCDD, and repeatability < 70%. Based on the data obtained, the micro-EROD assay is the most favorable bioanalytical tool, and via a micro-EROD-based limit value, it would allow for the assessment of sediment DLC concentrations; thus, it could be considered for the implementation into testing and management guidelines for dredged materials.
Assuntos
Dioxinas/toxicidade , Monitoramento Ambiental/métodos , Sedimentos Geológicos/análise , Poluentes Químicos da Água/toxicidade , Animais , Bioensaio , Linhagem Celular , Citocromo P-450 CYP1A1/metabolismo , Dioxinas/análise , Proteínas de Peixes/metabolismo , Alemanha , Oncorhynchus mykiss , Ratos , Receptores de Hidrocarboneto Arílico/metabolismo , Reprodutibilidade dos Testes , RiosRESUMO
A critical review of the last 25 years of dioxin policy in the Elbe river catchment is presented along seven main theses of the River Basin Community (RBC)-Elbe background document "Pollutants" for the Management Plan 2016-2021. In this period, polychlorinated dibenzodioxins/-furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs) will play a major role: (i) as new priority substances for which environmental quality standards (EQSs) need to be derived (Directive 2013/39/EC); (ii) in the search for innovative solutions in sediment remediation (i.e., respecting the influence of mechanical processes; Flood Risk Directive 2007/60/EC); and (iii) as indicators at the land-sea interface (Marine Strategy Framework Directive 2008/56/EC). In the Elbe river catchment, aspects of policy and science are closely connected, which became particularly obvious in a classic example of dioxin hot spot contamination, the case of the Spittelwasser creek. Here, the "source-first principle" of the first cycle of the European Water Framework Directive (WFD) had to be confirmed in a controversy on the dioxin hot spots with Saxony-Anhalt's Agency for Contaminated Sites (LAF). At the Spittelwasser site, the move from "inside the creek" to "along the river banks" goes parallel to a general paradigm shift in retrospective risk assessment frameworks and remediation techniques for organic chemicals (Ortega-Calvo et al. 2015). With respect to dioxin, large-scale stabilization applying activated carbon additions is particularly promising. Another important aspect is the assessment of the ecotoxicology of dioxins and dl- PCBs in context of sediment mobility and flood risk assessment, which has been studied in the project framework FloodSearch. Currently, the quality goals of the WFD to reach a "good chemical status" are not met in many catchment areas because substances such as mercury do and others probably will (PCDD/Fs and dl-PCB) exceed biota-EQS values catchment area-wide. So far, relating biota-EQS values to sediment-EQSs is not possible. To overcome these limitations, the DioRAMA project was initiated, which has led to improved approaches for the assessment of dioxin-contaminated sediment using in vitro bioassays and to a robust dataset on the interrelation between dioxins and dioxin-like compounds in sediments and biota.
RESUMO
BACKGROUND: Both frequency and intensity of flood events are expected to increase as a result of global climate change in the upcoming decades, potentially resulting in increased re-suspension of sediments in fluvial systems. Contamination of these re-suspended sediments with legacy contaminants, including dioxins and dioxin-like compounds (DLCs), as well as polycyclic aromatic hydrocarbons (PAHs) is of great ecotoxicological concern. DLCs, and to some extent also PAHs, exhibit their toxicity through activation of the aryl hydrocarbon receptor (AhR). However, interactions of DLCs with pathways other than those known to be mediated through the AhR are not fully understood to date. METHODS: This study aimed to investigate molecular and biochemical effects in roach (Rutilus rutilus) during a 10 days exposure to suspensions of three natural sediments that differed in the level of DLC contamination. Concentrations of biliary PAH metabolites and hepatic 7-ethoxyresorufin-O-deethylase activity were quantified in exposed fish. Furthermore, the abundance of transcripts of several genes related to energy metabolism, response to oxidative stress, and apoptosis, as well as cytochrome P450 1A (cyp1a) was quantified. RESULTS: Biliary PAH metabolites and activation of the AhR were confirmed as suitable early warning biomarkers of exposure to suspended sediments containing DLCs and PAHs that corresponded well with analytically determined concentrations of those contaminants. Although the abundances of transcripts of superoxide dismutase (sod), protein kinase c delta (pkcd), and ATP-binding cassette transporter c9 (abcc9) were altered by the treatment compared with unexposed control fish, none of these showed a time- or concentration-dependent response. The abundance of transcripts of pyruvate carboxylase (pc) and transferrin variant d (tfd) remained unaltered by the treatments. CONCLUSIONS: We have shown that contaminated sediments can become a risk for fish during re-suspension events (e.g., flooding and dredging). We have also demonstrated that roach, which are native to most European freshwater systems, are suitable sentinel species due to their great sensitivity and ecological relevance. Roach may be particularly suitable in future field studies to assess the toxicological concerns associated with the release of DLCs and PAHs during sediment re-suspension.
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There is significant concern regarding the contamination of riverine sediments with dioxins and dioxin-like compounds (DLCs), including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and some polycyclic aromatic hydrocarbons (PAHs). The majority of studies investigating the ecotoxicology of DLCs in fish have focused on a few standard model species. However, there is significant uncertainty as to whether these model species are representative of native river fish, particularly in Europe. In this study, the transcriptional responses following exposure to equipotent concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 156 or the dioxin-like PAH, benzo[k]fluoranthene (BkF), were investigated in juvenile roach (Rutilus rutilus), a fish species that constitutes a large proportion of the fish biomass in freshwater bodies throughout Europe. To this end, RNA sequencing analysis was used to comprehensively characterise the molecular mechanisms and pathways of toxicity of these DLCs. Whole transcriptome analyses using ClueGO software revealed that DLCs have the potential to disrupt a number of important processes, including energy metabolism, oogenesis, the immune system, apoptosis and the response to oxidative stress. However, despite using equipotent concentrations, there was very little conservation of the transcriptional responses observed in fish exposed to different DLCs. TCDD provoked significant specific changes in the levels of transcripts related to immunotoxicity and carbohydrate metabolism, while PCB 156 caused virtually no specific effects. Exposure to BkF affected the most diverse suite of molecular functions and biological processes, including blood coagulation, oxidative stress responses, unspecific responses to organic or inorganic substances/stimuli, cellular redox homeostasis and specific receptor pathways. To our knowledge, this is the first study of the transcriptome-wide effects of different classes of DLCs in fish. These findings represent an important step towards describing complete toxicity pathways of DLCs, which will be important in the context of informing risk assessments of DLC toxicity in native fish species.
Assuntos
Benzofuranos/toxicidade , Cyprinidae/fisiologia , Dioxinas/toxicidade , Poluentes Ambientais/toxicidade , Expressão Gênica/efeitos dos fármacos , Animais , Dibenzofuranos Policlorados , Europa (Continente) , Perfilação da Expressão GênicaRESUMO
To examine the uptake of dioxin-like compounds (DLCs), common roaches (Rutilus rutilus) were exposed for 28 days to differently contaminated sediments from two major European rivers in a purpose-built facility. Dietary transfer of DLCs was investigated by exposing fish to sediments inoculated or non-inoculated with black worms (Lumbriculus variegatus). Dioxin-like polychlorinated biphenyls (DL-PCBs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), measured via high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) in sediments and whole fish, were used to calculate toxicity equivalent quotients (TEQs). TEQs were compared with biological toxicity equivalent quotients (BEQs) determined via the 7-ethoxyresorufin-O-deethylase (EROD) assay, performed with mammalian (H4IIE) and fish (RTL-W1) liver cell lines. TEQs and BEQs indicated an uptake of sediment-borne DLCs by roach, which was independent of sediment contamination levels, but rather reflected sediment-specific characteristics. For most sediment treatments, DLC uptake did not increase with time. Highest congener-specific uptake (DL-PCB 123) was 10-fold compared to control. Exposure to worm-inoculated sediment of highest overall DLC contamination caused a 2-fold (TEQ and H4IIE BEQ) greater uptake of DLCs by fish compared to the respective non-inoculated treatment. H4IIE cells showed the greatest sensitivity (0.37 ± 0.25 pM TCDD) and the strongest correlation with TEQs (r (2) = 0.79), hence, they seem to be best suited for DLC screening of sediments and biota, amended by compound-specific instrumental analysis if required.
Assuntos
Dibenzofuranos/farmacocinética , Dioxinas/farmacocinética , Bifenilos Policlorados/farmacocinética , Poluentes Químicos da Água/farmacocinética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cyprinidae/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Dibenzofuranos/toxicidade , Dieta , Dioxinas/toxicidade , Sedimentos Geológicos/química , Hepatócitos/metabolismo , Bifenilos Policlorados/toxicidade , Ratos , Poluentes Químicos da Água/toxicidadeRESUMO
Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events. Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Sedimentos Geológicos/química , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Oncorhynchus mykiss/fisiologia , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Alemanha , Músculo Esquelético/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/análise , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Rios/química , Poluentes Químicos da Água/metabolismoRESUMO
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.
Assuntos
Citocromo P-450 CYP1A1/biossíntese , Dioxinas/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Fluorescência , Fluorometria/métodos , Oxazinas/metabolismo , RatosRESUMO
Use of in vitro assays as screening tool to characterize contamination of a variety of environmental matrices has become an increasingly popular and powerful toolbox in the field of environmental toxicology. While bioassays cannot entirely substitute analytical methods such as gas chromatography-mass spectrometry (GC-MS), the increasing improvement of cell lines and standardization of bioassay procedures enhance their utility as bioanalytical pre-screening tests prior to more targeted chemical analytical investigations. Dioxin-receptor-based assays provide a holistic characterization of exposure to dioxin-like compounds (DLCs) by integrating their overall toxic potential, including potentials of unknown DLCs not detectable via e.g. GC-MS. Hence, they provide important additional information with respect to environmental risk assessment of DLCs. This review summarizes different in vitro bioassay applications for detection of DLCs and considers the comparability of bioassay and chemical analytically derived toxicity equivalents (TEQs) of different approaches and various matrices. These range from complex samples such as sediments through single reference to compound mixtures. A summary of bioassay derived detection limits (LODs) showed a number of current bioassays to be equally sensitive as chemical methodologies, but moreover revealed that most of the bioanalytical studies conducted to date did not report their LODs, which represents a limitation with regard to low potency samples.
Assuntos
Bioensaio/métodos , Carcinógenos/metabolismo , Dioxinas/metabolismo , Animais , Cromatografia Gasosa-Espectrometria de Massas , Limite de Detecção , RatosRESUMO
As a consequence of global climate change, we will be likely facing an increasing frequency and intensity of flood events. Thus, the ecotoxicological relevance of sediment re-suspension is of growing concern. It is vital to understand contaminant uptake from suspended sediments and relate it to effects in aquatic biota. Here we report on a computational study that utilizes a physiologically based toxicokinetic model to predict uptake, metabolism and excretion of sediment-borne pyrene in rainbow trout (Oncorhynchus mykiss). To this end, data from two experimental studies were compared with the model predictions: (a) batch re-suspension experiments with constant concentration of suspended particulate matter at two different temperatures (12 and 24°C), and (b) simulated flood events in an annular flume. The model predicted both the final concentrations and the kinetics of 1-hydroxypyrene secretion into the gall bladder of exposed rainbow trout well. We were able to show that exhaustive exercise during exposure in simulated flood events can lead to increased levels of biliary metabolites and identified cardiac output and effective respiratory volume as the two most important factors for contaminant uptake. The results of our study clearly demonstrate the relevance and the necessity to investigate uptake of contaminants from suspended sediments under realistic exposure scenarios.
Assuntos
Inundações , Modelos Biológicos , Oncorhynchus mykiss/metabolismo , Pirenos/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Bile/química , Débito Cardíaco/efeitos dos fármacos , Sedimentos Geológicos/química , Atividade Motora , Pirenos/análise , Pirenos/toxicidade , Medição de Risco , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
In this study, in vitro bioassays were performed to assess the ecotoxicological potential of sediments from Yangtze River estuary. The cytotoxicity and aryl hydrocarbon receptor (AhR)-mediated toxicity of sediment extracts with rainbow trout (Oncorhynchus mykiss) liver cells were determined by neutral red retention and 7-ethoxyresorufin-O-deethylase assays. The cytotoxicity and AhR-mediated activity of sediments from the Yangtze River estuary ranged from low level to moderate level compared with the ecotoxicity of sediments from other river systems. However, Yangtze River releases approximately 14 times greater water discharge compared with Rhine, a major river in Europe. Thus, the absolute pollution mass transfer of Yangtze River may be detrimental to the environmental quality of estuary and East China Sea. Effect-directed analysis was applied to identify substances causing high dioxin-like activities. To identify unknown substances contributing to dioxin-like potencies of whole extracts, we fractionated crude extracts by open column chromatography. Non-polar paraffinic components (F1), weakly and moderately polar components (F2), and highly polar substances (F3) were separated from each crude extract of sediments. F2 showed the highest dioxin-like activities. Based on the results of mass balance calculation of chemical toxic equivalent concentrations (TEQs), our conclusion is that priority polycyclic aromatic hydrocarbons indicated a low portion of bio-TEQs ranging from 1% to 10% of crude extracts. Further studies should be conducted to identify unknown pollutants.