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Synthetic organic chemicals, including pesticides, pharmaceuticals, and industrial compounds, pose a growing threat to marine ecosystems. Despite their potential impact, data on the co-occurrence of these contaminants in multiple compartments, including surface water, bottom water, porewater, and sediment in the marine environment remains limited. Such information is critical for assessing coastal chemical status, establishing environmental quality benchmarks, and conducting comprehensive environmental risk assessments. In this study, we describe a multifaceted monitoring campaign targeting pesticides, pharmaceuticals, surfactants, additives, and plasticizers among other synthetic chemicals in four sampling sites. One site was located in the small Coliumo bay affected by urban settlements and tourism in central-south and additionally, we sampled three sites, Caucahue Channel, affected by urban settlements and salmon farming in northern Patagonia in Chile. Surface water, bottom water, porewater, and adjacent sediment samples were collected for target screening analysis in LC- and GC-HRMS platforms. Our results show the detection of up to 83 chemicals in surface water, 71 in bottom water, 101 in porewater, and 244 in sediments. To enhance data utility and reuse potential, we provide valuable information on the mode of action and molecular targets of the identified chemicals. This comprehensive dataset contributes to defining pollution fingerprints in coastal areas of the Global South, including remote regions in Patagonia. It serves as a critical resource for future research including marine chemical risk assessment, policymaking, and the advancement of environmental protection in these regions.
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A growing body of scientific literature stresses the need to advance current environmental risk assessment (ERA) methodologies and associated regulatory frameworks to better address the landscape-scale and long-term impact of pesticide use on biodiversity and the ecosystem. Moreover, more collaborative and integrative approaches are needed to meet sustainability goals. The One Health approach is increasingly applied by the European Food Safety Authority (EFSA) to support the transition towards safer, healthier and more sustainable food. To this end, EFSA commissioned the development of a roadmap for action to establish a European Partnership for next-generation, systems-based Environmental Risk Assessment (PERA). Here, we summarise the main conclusions and recommendations reported in the 2022 PERA Roadmap. This roadmap highlights that fragmentation of data, knowledge and expertise across regulatory sectors results in suboptimal processes and hinders the implementation of integrative ERA approaches needed to better protect the environment. To advance ERA, we revisited the underlying assumptions of the current ERA paradigm; that chemical risks are generally assessed and managed in isolation with a substance-by-substance, realistic worst-case and tiered approach. We suggest optimising the use of the vast amount of information and expertise available with pesticides as a pilot area. It is recommended to as soon as possible adopt a systems-based approach, i.e. within the current regulatory framework, to spark a step-wise transition towards an ERA framed at a system level of ecological and societal relevance. Tangible systems-based and integrative steps are available. For instance, the rich sources of existing data for prospective and retrospective ERA of pesticides could be used to reality-benchmark existing and new ERA methods. To achieve these goals, collaboration among stakeholders across scientific disciplines and regulatory sectors must be strengthened.
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Plaguicidas , Medición de Riesgo , Europa (Continente) , Análisis de Sistemas , Proyectos Piloto , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/análisis , Política AmbientalRESUMEN
Environmental hazard assessments are reliant on toxicity data that cover multiple organism groups. Generating experimental toxicity data is, however, resource-intensive and time-consuming. Computational methods are fast and cost-efficient alternatives, but the low accuracy and narrow applicability domains have made their adaptation slow. Here, we present a AI-based model for predicting chemical toxicity. The model uses transformers to capture toxicity-specific features directly from the chemical structures and deep neural networks to predict effect concentrations. The model showed high predictive performance for all tested organism groups-algae, aquatic invertebrates and fish-and has, in comparison to commonly used QSAR methods, a larger applicability domain and a considerably lower error. When the model was trained on data with multiple effect concentrations (EC50/EC10), the performance was further improved. We conclude that deep learning and transformers have the potential to markedly advance computational prediction of chemical toxicity.
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Organismos Acuáticos , Suministros de Energía Eléctrica , Animales , Redes Neurales de la ComputaciónRESUMEN
Environmental risk assessments strategies that account for the complexity of exposures are needed in order to evaluate the toxic pressure of emerging chemicals, which also provide suggestions for risk mitigation and management, if necessary. Currently, most studies on the co-occurrence and environmental impacts of chemicals of emerging concern (CECs) are conducted in countries of the Global North, leaving massive knowledge gaps in countries of the Global South. In this study, we implement a multi-scenario risk assessment strategy to improve the assessment of both the exposure and hazard components in the chemical risk assessment process. Our strategy incorporates a systematic consideration and weighting of CECs that were not detected, as well as an evaluation of the uncertainties associated with Quantitative Structure-Activity Relationships (QSARs) predictions for chronic ecotoxicity. Furthermore, we present a novel approach to identifying mixture risk drivers. To expand our knowledge beyond well-studied aquatic ecosystems, we applied this multi-scenario strategy to the River Aconcagua basin of Central Chile. The analysis revealed that the concentrations of CECs exceeded acceptable risk thresholds for selected organism groups and the most vulnerable taxonomic groups. Streams flowing through agricultural areas and sites near the river mouth exhibited the highest risks. Notably, the eight risk drivers among the 153 co-occurring chemicals accounted for 66-92 % of the observed risks in the river basin. Six of them are pesticides and pharmaceuticals, chemical classes known for their high biological activity in specific target organisms.
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Monitoreo del Ambiente , Contaminantes Químicos del Agua , Contaminantes Químicos del Agua/análisis , Ecosistema , Ríos/química , Chile , Medición de RiesgoRESUMEN
Pharmaceuticals and personal care products (PPCPs) occur as variable mixtures in surface waters receiving discharges of human and animal wastes. A key question identified a decade ago is how to assess the effects of long-term exposures of these PPCP mixtures on nontarget organisms. We review the recent progress made on assessing the aquatic ecotoxicity of PPCP mixtures-with a focus on active pharmaceutical ingredients-and the challenges and research needs that remain. New knowledge has arisen from the use of whole-mixture testing combined with component-based approaches, and these studies show that mixtures often result in responses that meet the concentration addition model. However, such studies have mainly been done on individual species over shorter time periods, and longer-term, multispecies assessments remain limited. The recent use of targeted and nontargeted gene analyses has improved our understanding of the diverse pathways that are impacted, and there are promising new "read-across" methods that use mammalian data to predict toxicity in wildlife. Risk assessments remain challenging given the paucity of ecotoxicological and exposure data on PPCP mixtures. As such, the assessment of PPCP mixtures in aquatic environments should remain a priority given the potential for additive-as well as nontarget-effects in nontarget organisms. In addition, we need to improve our understanding of which species, life stages, and relevant endpoints are most sensitive to which types of PPCP mixtures and to expand our knowledge of environmental PPCP levels in regions of the globe that have been poorly studied to date. We recommend an increased use of new approach methodologies, in particular "omics," to advance our understanding of the molecular mechanics of mixture effects. Finally, we call for systematic research on the role of PPCP mixtures in the development of antimicrobial resistance. Environ Toxicol Chem 2024;43:549-558. © 2023 SETAC.
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Cosméticos , Contaminantes Químicos del Agua , Animales , Humanos , Ecosistema , Ecotoxicología , Medición de Riesgo , Preparaciones Farmacéuticas , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/análisis , Monitoreo del Ambiente/métodos , Cosméticos/análisis , MamíferosRESUMEN
Environmental exposure data are used by decision-makers to assess environmental risks and implement actions to mitigate risks from contaminants. The first article in this series summarized the available evaluation schemes for environmental exposure data, of which there are few compared to those available for environmental hazard data. The second article covered the assessment of the reliability of environmental exposure data sets under the Criteria for the Reporting and Evaluation of Exposure Data (CREED). The aim of this article is to provide an overview and practical guidance on the relevance assessment in the context of the CREED approach for evaluating exposure monitoring data sets. Systematically considering relevance is critical for both evaluating existing data sets and for optimizing the design of new monitoring studies. Relevance is defined here as the degree of suitability or appropriateness of a data set to address a specific purpose or to answer the questions that have been defined by the assessor or for those generating exposure data. The purpose definition will be the foundation for the relevance assessment, to clarify how the assessor should rate the assessment criteria (fully met, partly met, not met/inappropriate, not reported, not applicable). This will provide transparency for anyone reviewing the outcomes. An explicit gap analysis (i.e., an articulation of the data set limitations for the stated purpose) is an important outcome of the relevance assessment. The relevance evaluation approach is demonstrated with three case studies, all relating to the freshwater aquatic environment, where the data sets are scored as relevant with or without restrictions, not relevant, or not assignable. The case studies represent both organic and inorganic constituents, and have different data characteristics (e.g., percentage of censored data, sampling frequencies, relation to supporting parameters). Integr Environ Assess Manag 2024;20:1004-1018. © 2023 SETAC.
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Exposición a Riesgos Ambientales , Monitoreo del Ambiente , Monitoreo del Ambiente/métodos , Exposición a Riesgos Ambientales/estadística & datos numéricos , Medición de Riesgo/métodos , Contaminantes Ambientales/análisis , Reproducibilidad de los ResultadosRESUMEN
There are more than 3,500 active pharmaceutical ingredients (APIs) on the global market for human and veterinary use. Residues of these APIs eventually reach the aquatic environment. Although an environmental risk assessment (ERA) for marketing authorization applications of medicinal products is mandatory in the European Union since 2006, an ERA is lacking for most medicines approved prior to 2006 (legacy APIs). Since it is unfeasible to perform extensive ERA tests for all these legacy APIs, there is a need for prioritization of testing based on the limited data available. Prioritized APIs can then be further investigated to estimate their environmental risk in more detail. In this study, we prioritized more than 1,000 APIs used in Europe based on their predicted risk for aquatic freshwater ecosystems. We determined their risk by combining an exposure estimate (Measured or Predicted Environmental Concentration; MEC or PEC, respectively) with a Predicted No Effect Concentration (PNEC). We developed several procedures to combine the limited empirical data available with in silico data, resulting in multiple API rankings varying in data needs and level of conservativeness. In comparing empirical with in silico data, our analysis confirmed that the PEC estimated with the default parameters used by the European Medicines Agency often - but not always - represents a worst-case scenario. Comparing the ecotoxicological data for the three main taxonomic groups, we found that fish represents the most sensitive species group for most of the APIs in our list. We furthermore show that the use of in silico tools can result in a substantial underestimation of the ecotoxicity of APIs. After combining the different exposure and effect estimates into four risk rankings, the top-ranking APIs were further screened for availability of ecotoxicity data in data repositories. This ultimately resulted in the prioritization of 15 APIs for further ecotoxicological testing and/or exposure assessment.
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Monitoreo del Ambiente , Contaminantes Químicos del Agua , Animales , Humanos , Monitoreo del Ambiente/métodos , Ecosistema , Medición de Riesgo/métodos , Peces , Preparaciones Farmacéuticas , Contaminantes Químicos del Agua/análisisRESUMEN
Pollution by chemicals and waste impacts human and ecosystem health on regional, national, and global scales, resulting, together with climate change and biodiversity loss, in a triple planetary crisis. Consequently, in 2022, countries agreed to establish an intergovernmental science-policy panel (SPP) on chemicals, waste, and pollution prevention, complementary to the existing intergovernmental science-policy bodies on climate change and biodiversity. To ensure the SPP's success, it is imperative to protect it from conflicts of interest (COI). Here, we (i) define and review the implications of COI, and its relevance for the management of chemicals, waste, and pollution; (ii) summarize established tactics to manufacture doubt in favor of vested interests, i.e., to counter scientific evidence and/or to promote misleading narratives favorable to financial interests; and (iii) illustrate these with selected examples. This analysis leads to a review of arguments for and against chemical industry representation in the SPP's work. We further (iv) rebut an assertion voiced by some that the chemical industry should be directly involved in the panel's work because it possesses data on chemicals essential for the panel's activities. Finally, (v) we present steps that should be taken to prevent the detrimental impacts of COI in the work of the SPP. In particular, we propose to include an independent auditor's role in the SPP to ensure that participation and processes follow clear COI rules. Among others, the auditor should evaluate the content of the assessments produced to ensure unbiased representation of information that underpins the SPP's activities.
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Conflicto de Intereses , Ecosistema , Humanos , Contaminación Ambiental , BiodiversidadRESUMEN
Empirical and in silico data on the aquatic ecotoxicology of 2697 organic chemicals were collected in order to compile a dataset for assessing the predictive power of current Quantitative Structure Activity Relationship (QSAR) models and software platforms. This document presents the dataset and the data pipeline for its creation. Empirical data were collected from the US EPA ECOTOX Knowledgebase (ECOTOX) and the EFSA (European Food Safety Authority) report "Completion of data entry of pesticide ecotoxicology Tier 1 study endpoints in a XML schema - database". Only data for OECD recommended algae, daphnia and fish species were retained. QSAR toxicity predictions were calculated for each chemical and each of six endpoints using ECOSAR, VEGA and the Toxicity Estimation Software Tool (T.E.S.T.) platforms. Finally, the dataset was amended with SMILES, InChIKey, pKa and logP collected from webchem and PubChem.
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Plastics are produced with a staggering array of chemical compounds, with many being known to possess hazardous properties, and others lacking comprehensive hazard data. Furthermore, non-intentionally added substances can contaminate plastics at various stages of their lifecycle, resulting in recycled materials containing an unknown number of chemical compounds at unknown concentrations. While some national and regional regulations exist for permissible concentrations of hazardous chemicals in specific plastic products, less than 1 % of plastics chemicals are subject to international regulation [1]. There are currently no policies mandating transparent reporting of chemicals throughout the plastics value chain or comprehensive monitoring of chemicals in recycled materials. The dataset presented here provides the chemical analysis of 28 samples of recycled High-Density Polyethylene (HDPE) pellets obtained from various regions of the Global South, along with a reference sample of virgin HDPE. The analysis comprises both Target and Non-Targeted Screening approaches, employing Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS) and Gas Chromatography-High-Resolution Mass Spectrometry (GC-HRMS). In total, 491 organic compounds were detected and quantified, with an additional 170 compounds tentatively annotated. These compounds span various classes, including pesticides, pharmaceuticals, industrial chemicals, plastic additives. The results highlight the prevalence of certain chemicals, such as N-ethyl-o-Toluesulfonamide, commonly used in HDPE processing, found in high concentrations. The paper provides a dataset advancing knowledge of the complex chemical composition associated with recycled plastics.
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Chemical pollution caused by synthetic organic chemicals at low concentrations in the environment poses a growing threat to the ecological status of aquatic ecosystems. These chemicals are regularly released into surface waters through both treated and untreated effluents from wastewater treatment plants (WWTPs), agricultural runoff, and industrial discharges. Consequently, they accumulate in surface waters, distribute amongst environmental compartments according to their physicochemical properties, and cause adverse effects on aquatic organisms. Unfortunately, there is a lack of data regarding the occurrence of synthetic organic chemicals, henceforth micropollutants, in South American freshwater ecosystems, especially in Chile. To address this research gap, we present a comprehensive dataset comprising concentrations of 153 emerging chemicals, including pesticides, pharmaceutical and personal care products (PPCPs), surfactants, and industrial chemicals. These chemicals were found to co-occur in surface waters within Central Chile, specifically in the River Aconcagua Basin. Our sampling strategy involved collecting surface water samples from streams and rivers with diverse land uses, such as agriculture, urban areas, and natural reserves. For sample extraction, we employed an on-site large-volume solid phase extraction (LVSPE) device. The resulting environmental extracts were then subjected to wide-scope chemical target screening using gas chromatography and liquid chromatography high-resolution mass spectrometry (GC- and LC-HRMS). The dataset we present holds significant value in assessing the chemical status of water bodies. It enables comparative analysis of pollution fingerprints associated with emerging chemicals across different freshwater systems. Moreover, the data can be reused for environmental risk assessment studies. Its utilisation will contribute to a better understanding of the impact and extent of chemical pollution in aquatic ecosystems, facilitating the development of effective mitigation strategies.
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Food contact materials (FCMs) and food contact articles are ubiquitous in today's globalized food system. Chemicals migrate from FCMs into foodstuffs, so called food contact chemicals (FCCs), but current regulatory requirements do not sufficiently protect public health from hazardous FCCs because only individual substances used to make FCMs are tested and mostly only for genotoxicity while endocrine disruption and other hazard properties are disregarded. Indeed, FCMs are a known source of a wide range of hazardous chemicals, and they likely contribute to highly prevalent non-communicable diseases. FCMs can also include non-intentionally added substances (NIAS), which often are unknown and therefore not subject to risk assessment. To address these important shortcomings, we outline how the safety of FCMs may be improved by (1) testing the overall migrate, including (unknown) NIAS, of finished food contact articles, and (2) expanding toxicological testing beyond genotoxicity to multiple endpoints associated with non-communicable diseases relevant to human health. To identify mechanistic endpoints for testing, we group chronic health outcomes associated with chemical exposure into Six Clusters of Disease (SCOD) and we propose that finished food contact articles should be tested for their impacts on these SCOD. Research should focus on developing robust, relevant, and sensitive in-vitro assays based on mechanistic information linked to the SCOD, e.g., through Adverse Outcome Pathways (AOPs) or Key Characteristics of Toxicants. Implementing this vision will improve prevention of chronic diseases that are associated with hazardous chemical exposures, including from FCMs.
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Contaminación de Alimentos , Enfermedades no Transmisibles , Humanos , Contaminación de Alimentos/análisis , Salud Pública , Embalaje de Alimentos , Alimentos , Sustancias Peligrosas/toxicidadRESUMEN
Climate change, biodiversity loss, and chemical pollution are planetary-scale emergencies requiring urgent mitigation actions. As these "triple crises" are deeply interlinked, they need to be tackled in an integrative manner. However, while climate change and biodiversity are often studied together, chemical pollution as a global change factor contributing to worldwide biodiversity loss has received much less attention in biodiversity research so far. Here, we review evidence showing that the multifaceted effects of anthropogenic chemicals in the environment are posing a growing threat to biodiversity and ecosystems. Therefore, failure to account for pollution effects may significantly undermine the success of biodiversity protection efforts. We argue that progress in understanding and counteracting the negative impact of chemical pollution on biodiversity requires collective efforts of scientists from different disciplines, including but not limited to ecology, ecotoxicology, and environmental chemistry. Importantly, recent developments in these fields have now enabled comprehensive studies that could efficiently address the manifold interactions between chemicals and ecosystems. Based on their experience with intricate studies of biodiversity, ecologists are well equipped to embrace the additional challenge of chemical complexity through interdisciplinary collaborations. This offers a unique opportunity to jointly advance a seminal frontier in pollution ecology and facilitate the development of innovative solutions for environmental protection.
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Ecosistema , Contaminación Ambiental , Biodiversidad , Ecología , Conservación de los Recursos Naturales , Cambio ClimáticoRESUMEN
While chemicals are vital to modern society through materials, agriculture, textiles, new technology, medicines, and consumer goods, their use is not without risks. Unfortunately, our resources seem inadequate to address the breadth of chemical challenges to the environment and human health. Therefore, it is important we use our intelligence and knowledge wisely to prepare for what lies ahead. The present study used a Delphi-style approach to horizon-scan future chemical threats that need to be considered in the setting of chemicals and environmental policy, which involved a multidisciplinary, multisectoral, and multinational panel of 25 scientists and practitioners (mainly from the United Kingdom, Europe, and other industrialized nations) in a three-stage process. Fifteen issues were shortlisted (from a nominated list of 48), considered by the panel to hold global relevance. The issues span from the need for new chemical manufacturing (including transitioning to non-fossil-fuel feedstocks); challenges from novel materials, food imports, landfills, and tire wear; and opportunities from artificial intelligence, greater data transparency, and the weight-of-evidence approach. The 15 issues can be divided into three classes: new perspectives on historic but insufficiently appreciated chemicals/issues, new or relatively new products and their associated industries, and thinking through approaches we can use to meet these challenges. Chemicals are one threat among many that influence the environment and human health, and interlinkages with wider issues such as climate change and how we mitigate these were clear in this exercise. The horizon scan highlights the value of thinking broadly and consulting widely, considering systems approaches to ensure that interventions appreciate synergies and avoid harmful trade-offs in other areas. We recommend further collaboration between researchers, industry, regulators, and policymakers to perform horizon scanning to inform policymaking, to develop our ability to meet these challenges, and especially to extend the approach to consider also concerns from countries with developing economies. Environ Toxicol Chem 2023;42:1212-1228. © 2023 Crown copyright and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.
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Inteligencia Artificial , Contaminación Ambiental , Humanos , Ecotoxicología , Agricultura , Europa (Continente)RESUMEN
Chemicals emitted to the environment affect ecosystem health from local to global scale, and reducing chemical impacts has become an important element of European and global sustainability efforts. The present work advances ecotoxicity characterization of chemicals in life cycle impact assessment by proposing recommendations resulting from international expert workshops and work conducted under the umbrella of the UNEP-SETAC Life Cycle Initiative in the GLAM project (Global guidance on environmental life cycle impact assessment indicators). We include specific recommendations for broadening the assessment scope through proposing to introduce additional environmental compartments beyond freshwater and related ecotoxicity indicators, as well as for adapting the ecotoxicity effect modelling approach to better reflect environmentally relevant exposure levels and including to a larger extent chronic test data. As result, we (1) propose a consistent mathematical framework for calculating freshwater ecotoxicity characterization factors and their underlying fate, exposure and effect parameters; (2) implement the framework into the USEtox scientific consensus model; (3) calculate characterization factors for chemicals reported in an inventory of a life cycle assessment case study on rice production and consumption; and (4) investigate the influence of effect data selection criteria on resulting indicator scores. Our results highlight the need for careful interpretation of life cycle assessment impact scores in light of robustness of underlying species sensitivity distributions. Next steps are to apply the recommended characterization framework in additional case studies, and to adapt it to soil, sediment and the marine environment. Our framework is applicable for evaluating chemicals in life cycle assessment, chemical and environmental footprinting, chemical substitution, risk screening, chemical prioritization, and comparison with environmental sustainability targets.
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Ecosistema , Agua Dulce , Agua Dulce/química , Modelos TeóricosRESUMEN
Regulating chemical mixtures is a complex scientific and policy task. The aim of this study was to investigate typical mixtures and their potential risks based on internal exposure levels in the European population. Based on human biomonitoring (HBM) data made available via the HBM4EU project, we derived generic mixtures representative of a median (P50) and a worst-case scenario (P95) for adults and children. We performed a mixture risk assessment based on HBM concentrations, health-based guidance values (HBGVs) as internal thresholds of concern, and the conservative assumption of concentration addition applied across different toxicological endpoints. Maximum cumulative ratios (MCRs) were calculated to characterize the mixture risk. The mixtures comprise 136 biomarkers for adults and 84 for children, although concentration levels could be quantified only for a fraction of these. Due to limited availability of HBGVs, the mixture risk was assessed for a subset of 20 substance-biomarker pairs for adults and 17 for children. The mixture hazard index ranged from 2.8 (P50, children) to 9.2 (P95, adults). Six to seven substances contributed to over 95% of the total risk. MCR values ranged between 2.6 and 5.5, which is in a similar range as in previous studies based on human external exposures assessments. The limited coverage of substances included in the calculations and the application of a hazard index across toxicological endpoints argue for caution in the interpretation of the results. Nonetheless the analyses of MCR and MAFceiling can help inform a possible mixture assessment factor (MAF) applicable to single substance risk assessment to account for exposure to unintentional mixtures.
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Monitoreo Biológico , Adulto , Niño , Humanos , Medición de Riesgo/métodosRESUMEN
Several contaminants exceed their environmental thresholds in Swedish marine surface waters. We ranked the toxicity-drivers among contaminants detected near heavy industry, based on toxicity to zooplankton, and identified di-butyl phthalate and sodium dodecyl sulphate as contaminants of concern. We tested their acute individual and mixture toxicity by assessing effects on mortality, biodiversity, algal growth, and copepod reproduction in natural mesozooplankton communities. The mixture effects were compared to Independent Action mixture model predictions. Egg production and algae growth were affected at 4 µmol/l DBP, and effects on mortality, algae growth and biodiversity were observed at 12 µmol/l SDS. The mixture (1 µmol/l DBP, 3 µmol/l SDS) affected all endpoints, and the observed effects were underestimated by 21% on average compared to predictions. We found that the successional trajectory in zooplankton communities was compound dependant, and that DBP and SDS are toxic to marine zooplankton, but at levels above measured environmental concentrations.
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The assessment of the exposure of aquatic wildlife to complex environmental mixtures of chemicals originating from both point and diffuse sources and evaluating the potential impact thereof constitutes a significant step towards mitigating toxic pressure and the improvement of ecological status. In the current proof-of-concept study, we demonstrate the potential of a novel Aggregated Biomarker Response (ABR) approach involving a comprehensive set of biomarkers to identify complex exposure and impacts on wild brown trout (Salmo trutta fario). Our scenario used a small lowland river in Germany (Holtemme river in the Elbe river catchment) impacted by two wastewater treatment plants (WWTP) and diffuse agricultural runoff as a case study. The trout were collected along a pollution gradient (characterised in a parallel study) in the river. Compared to fish from the reference site upstream of the first WWTP, the trout collected downstream of the WWTPs showed a significant increase in micronucleus formation, phase I and II enzyme activities, and oxidative stress parameters in agreement with increasing exposure to various chemicals. By integrating single biomarker responses into an aggregated biomarker response, the two WWTPs' contribution to the observed toxicity could be clearly differentiated. The ABR results were supported by chemical analyses and whole transcriptome data, which revealed alterations of steroid biosynthesis and associated pathways, including an anti-androgenic effect, as some of the key drivers of the observed toxicity. Overall, this combined approach of in situ biomarker responses complemented with molecular pathway analysis allowed for a comprehensive ecotoxicological assessment of fish along the river. This study provides evidence for specific hazard potentials caused by mixtures of agricultural and WWTP derived chemicals at sublethal concentrations. Using aggregated biomarker responses combined with chemical analyses enabled an evidence-based ranking of sites with different degrees of pollution according to toxic stress and observed effects.