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Around the world, many ambitious environmental conventions and regulations have been implemented over recent decades. Despite this, the environment is still deteriorating. An increase in the volume and diversity of chemicals is one of the main drivers of this deterioration, of which biodiversity loss is a telling indicator. In response to this situation, in October 2020, a chemicals strategy for sustainability (CSS) was published in the EU. The CSS is the first regional framework aiming to address chemical pollution in a holistic manner. The CSS covers the complete lifecycle of a chemical, including the design of better substances and remediation options, to remove chemicals from the environment. The strategy contains terms, such as a “toxic‐free environment,” for which no clear definition exists, potentially hampering the implementation of the CSS. In this paper, a definition for a “toxic‐free environment” is proposed on the basis of a survey and a discussion held at the 2020 SETAC Europe Annual Meeting. In addition, key issues that are absent from the CSS but are considered to be key for the realization of a toxic‐free environment are identified. To achieve the policy goals, it is recommended to align the definition of risk across the different chemical legislations, to establish a platform for open data and data sharing, and to increase the utility and use of novel scientific findings in policymaking, through the development of a strong science to regulation feedback mechanism and vice versa. The paper concludes that environmental scientists have the tools to address the key challenges presented in the CSS. However, an extra step is needed by both policymakers and scientists to develop methods, processes and tools, to increase the robustness and transparency of deliberation processes, and the utility of science. Integr Environ Assess Manag 2021;17:1105–1113. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC)., KEY POINTS A definition for a ‘toxic‐free environment’ is proposed based on an analysis of a survey and a discussion held at the 2020 SETAC Europe Annual Meeting.Key issues are identified based on discussions that are absent from the European Commission's chemicals strategy for sustainability but are considered to be key for the realisation of a toxic‐free environment.Scientists have the tools to address key environmental challenges, but an extra step is needed by both policymakers and scientists to make these applicable.

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 bymixtures 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.

Sve češće uočena izloženost pesticidima u okolišu ukazuje na potrebu procjene štetnih učinaka kako bi se razumjeli mehanizmi i umanjili negativni utjecaji. Istraživanja pokazuju da preporuka EU propisa na procjenu učinaka aktivnog sastojka, umjesto komercijalnog pripravka, može podcijeniti rizike i učinke izloženosti pesticidima na okoliš. Nadalje, važno je utvrditi treba li koristiti akutne i/ili kronične testove kako bi se precizno predvidjela toksičnost jer pesticid koji ne pokazuje akutne učinke i dalje bi mogao pokazati dugoročnu toksičnost. U ovom istraživanju testirano je djelovanje četiri komercijalna pripravka pesticida i njihovih aktivnih tvari, od čega su dva insekticida i dva herbicida. Akutna i kronična toksičnost na gujavice (Eisenia andrei) testirana je praćenjem mortaliteta, promjena u ponašanju (izbjegavanje), te mjerenjem različitih biomarkera i uspješnosti razmnožavanja. Usporedba akutne toksičnosti aktivnog sastojka s akutnom toksičnosti komercijalnog pripravka pokazala je nižu toksičnost aktivnog sastojka. Učinci uočeni na substaničnoj razini, zajedno s promjenama u ponašanju i reprodukciji (broju kokona), ukazuju na potencijalne štetne učinke istraživanih pesticida na gujavice u okolišu. Dobiveni rezultati upućuju na potrebu sveobuhvatnog ispitivanja toksičnosti pesticida uključujući istraživanje i komercijalnih pripravaka, kao i primjenu kratkoročnih i dugoročnih testova.

Around the world, many ambitious environmental conventions and regulations have been implemented over recent decades. Despite this, the environment is still deteriorating. An increase in the volume and diversity of chemicals is one of the main drivers of this deterioration, of which biodiversity loss is a telling indicator. In response to this situation, in October 2020, a chemicals strategy for sustainability (CSS) was published in the EU. The CSS is the first regional framework aiming to address chemical pollution in a holistic manner. The CSS covers the complete lifecycle of a chemical, including the design of better substances and remediation options, to remove chemicals from the environment. The strategy contains terms, such as a “toxic-free environment,” for which no clear definition exists, potentially hampering the implementation of the CSS. In this paper, a definition for a “toxic-free environment” is proposed on the basis of a survey and a discussion held at the 2020 SETAC Europe Annual Meeting. In addition, key issues that are absent from the CSS but are considered to be key for the realization of a toxic-free environment are identified. To achieve the policy goals, it is recommended to align the definition of risk across the different chemical legislations, to establish a platform for open data and data sharing, and to increase the utility and use of novel scientific findings in policymaking, through the development of a strong science to regulation feedback mechanism and vice versa. The paper concludes that environmental scientists have the tools to address the key challenges presented in the CSS. However, an extra step is needed by both policymakers and scientists to develop methods, processes and tools, to increase the robustness and transparency of deliberation processes, and the utility of science.

Around the world, many ambitious environmental conventions and regulations have been implemented over recent decades. Despite this, the environment is still deteriorating. An increase in the volume and diversity of chemicals is one of the main drivers of this deterioration, of which biodiversity loss is a telling indicator. In response to this situation, in October 2020, a chemicals strategy for sustainability (CSS) was published in the EU. The CSS is the first regional framework aiming to address chemical pollution in a holistic manner. The CSS covers the complete lifecycle of a chemical, including the design of better substances and remediation options, to remove chemicals from the environment. The strategy contains terms, such as a “toxic-free environment,” for which no clear definition exists, potentially hampering the implementation of the CSS. In this paper, a definition for a “toxic-free environment” is proposed on the basis of a survey and a discussion held at the 2020 SETAC Europe Annual Meeting. In addition, key issues that are absent from the CSS but are considered to be key for the realization of a toxic-free environment are identified. To achieve the policy goals, it is recommended to align the definition of risk across the different chemical legislations, to establish a platform for open data and data sharing, and to increase the utility and use of novel scientific findings in policymaking, through the development of a strong science to regulation feedback mechanism and vice versa. The paper concludes that environmental scientists have the tools to address the key challenges presented in the CSS. However, an extra step is needed by both policymakers and scientists to develop methods, processes and tools, to increase the robustness and transparency of deliberation processes, and the utility of science. Integr Environ Assess Manag 2021;17:1105–1113.

In the last decades the negative effects of pesticides on the environment have been increasingly observed worldwide. A widely known example is overall biodiversity decline often due to effects on non-target organisms. A vast biodiversity decline not only affects ecosystems and food chains but consequently also ecosystem services. In this study, we assessed the effects of four pesticides on earthworm (Eisenia andrei) in standardised soil: namely, insecticides thiacloprid (Calypso), esfenvalerate (Sumialfa) and herbicides prosulfocarb (Filon), and dimethenamid-p (Frontier). As current regulations only include standardised toxicity testing of the active substances, important data is missing on the different modes of action and possibly varying toxicity of commercial formulations. Hence, we investigated the effects of the commercially available pesticide formulations after 7, 14, and 28-day exposures. After determining sublethal concentrations in preliminary tests, a comprehensive test-battery, including biomarkers, fluorescence-based oxidative stress-related markers, multixenobiotic resistance and reproduction, has been used to determine the sublethal toxicity of the investigated pesticides. All pesticides showed effects on oxidative stress- related markers and multixenobiotic resistance. Prosulfocarb (Filon) and esfenvalerate (Sumialfa) both showed significant effects on multixenobiotic metabolism and cocoon production.

In recent years the debate about the usage of pesticides has increased widely. Biodiversity has not only been in observed in Germany but worldwide and is often linked to side-effects of pesticide usage. A vast biodiversity decline is not only a concern for entire ecosystems and food chains but could indirectly also impact ecosystem services. Due to e.g. rainfall pesticides can end up in aquatic systems and thus potentially cause negative effects on non- target organism. Improving the investigative tools for the assessment of pesticide effects is therefore necessary, to be able to understand their modes of action in more detail and extend the available data for pesticides already in use. An increased assessment often calls for a higher usage of test organisms, resulting in a dilemma as it has been an important aim to minimize the usage of laboratory animals. In this study, the effects of four pesticides on zebrafish larvae (Danio rerio) are assessed: namely, insecticides thiacloprid, esfenvalerate and herbicides prosulfocarb and dimethenamid-p. As current regulations only include standardized toxicity testing of the active substances, important data is missing on the different modes of action and possibly varying toxicity of commercial formulations and therefore both are investigated in this study. A comprehensive test-battery has been used and a workflow established to assess multiple sublethal endpoints, e.g. fluorescence-based methods oxidative stress and MXR activity detection as well as behavioral changes and biomarker measurements with a minimal number of test organisms.

The EU funded collaborative FP7 project SOLUTIONS searches for new and improved tools, models, and methods to produce consistent solutions for the large number of present and future emerging chemicals posing a risk to European water bodies with respect to ecosystems and human health. In the project framework, a special focus is put on the development, validation and documentation of a diagnostic toolbox to identify and quantify in situ impacts of mixtures of pollutants integrating over single organism, population and community levels. For this purpose, a battery of in situ biomarker and bioassay analyses in tissues of caged and free-living fish specimens from the River Danube were conducted in a weight- of-evidence approach. Gene expression analyses of selected endocrine disruption and stress representative genes, and antioxidant and biotransformation enzymes activity were investigated, to estimate the physiological status of the organisms exposed to chemical stress at pollution hot spots. Obtained results were evaluated against the chemical composition of the water, and the available results of in vitro bioassays. The study demonstrated a prominent role of physiological responses of fish as a part of a complex battery to (a) contribute to a better understanding of cause-effect relationships of exposure to key pollutants, and (b) provide useful data for prioritization of contaminants and contaminant mixtures in the aquatic environment. The ultimate goal is to support decisions in environmental and water policies regarding chemical regulation and abatement measures, and minimize ecological and human health risks.

Aquatic ecosystems are impacted by chemical pollution originating from various sources. Pesticides used in agriculture, forestry, households, etc. eventually enter aquatic ecosystems and can have hazardous effects on aquatic organisms. In aquatic ecotoxicological testing the zebrafish (Danio rerio) is commonly used as a model organism. The early life-stage test using the zebrafish embryo is one of the most widely used tools for assessment of toxicity and mechanisms of action of environmental pollutants. In the present study the acute toxicity of the insecticide diazinon and the herbicide diuron was assessed by conducting the Fish Embryo Toxicity (FET) test. From the results obtained in the FET test, sublethal concentrations for exposure experiments were derived. Subsequently, zebrafish embryos were exposed to the pesticides (0.02, 0.1, 0.5, 1, 2 mg L-1) for 48 and 96 h and enzymatic biomarkers (activities of AChE, CES, EROD, GST, CAT and GPx), fluorescent detection of GSH and gene expression (ache, ces2, cyp1a, gstp1, gpx1a, cat and gsr) was assessed. Both pesticides caused morphological aberrations such as pericardial and yolk sac edema, as well as reduction in spontaneous movements. Toxicity of the investigated pesticides was similar, with 96 h LC50 and EC50 values of 6.72±0.16 and 4.16±0.07 mg L-1 for diazinon, and 6.31±0.19 and 4.37±0.21 mg L-1 for diuron. The results from the exposure experiments showed significant changes in the measured biomarkers and indicated occurrence of oxidative stress. The measurement of endpoints at different organizational levels provided some new insights into the toxicity mechanisms of these pesticides to zebrafish.

Close collaboration with several case studies is one of the main advantages in terms of the realization of biomarker response analysis within the 7th EU RTD Framework Programme project SOLUTIONS (Solutions for present and emerging pollutants in land and water resource management). By using the infrastructure of these studies, biomarker monitoring approaches can be performed easily. Under the lead of the Helmholtz-Centre for Environmental Research the small river Holtemme was investigated in an interdisciplinary approach. The stream has its source in the Mountain Harz region (Germany) and flows through a number of small and medium- sized cities in Saxony-Anhalt. Due to its low water-quantity and several anthropogenic point sources the Holtemme is a very valuable model site to assess the impact of pollution induced biomarker responses in aquatic organisms. 10 individuals of brown trout (Salmo trutta) were collected at each of 4 different locations including a “natural” reference site. Locations were downstream waste water treatment plants (WWTPs) and one location in a more agriculturally influenced river section. The utilized biomarker battery covered a broad range of proven endpoints which have been employed in several previous studies: genotoxicity in peripheral erythrocytes (micronucleus test), biotransformation enzyme activity (EROD, GST, CES), oxidative stress (TBARS, CAT) and biomarker of exposure to organophosphate and carbamate pesticides (AChE). To reduce time and sample consumption all common cuvette analysis for these endpoints were adapted to 96-well plate assays. For most of the investigated biomarkers significantly elevated/lowered values for the urban and agriculture-affected sampling sites could be demonstrated in comparison to the reference site. However, the results indicated no similar trend for all biomarker responses along the longitudinal profile of the River Holtemme, but the individual biomarkers gave “hot-spots” of response. Correlation analysis with chemical data might help to identify presumed stressors related to the different observed effects.

Reactive toxicity is a first line response of cells upon exposure to toxic substances and part of the process that leads to cytotoxicity of contaminants. Detection of reactive toxicity in complex organisms can help to identify cell stress way before effects become visible on an organ or organism level. This allows for an early and sensitive identification of adverse effects. A zebrafish embryo assay that uses a fluorescent dye for microscopic detection of glutathion was adapted to a 96-well plate format to allow for quantification of reactive toxicity through fluorescence measurement with a multiwell plate reader. The assay was initially developed using tert-butyl hydroperoxide (t-BHP) as a reference substance, and then benchmark tested against the two pesticides diazinon and diuron. Long and short- term exposure scenarios and different protocols for the fluorescent staining were used. Hatched larvae (96 hpf) were then anesthetized, transferred to 96-well plates with conical wells, and fluorescence measured using a tailored detection method for a multiwell plate reader. Embryos were also inspected under a fluorescence microscope to qualitatively record locations and intensities of reactive toxicity in the individual specimen. Prior to the assay, lethal and sublethal effects of the pesticides on zebrafish embryos had been determined in a common FET test. We found that the assay is capable of detecting reactive toxicity of diazinon and diuron at concentrations as low as 0.1 mg/L and 0.02 mg/L, respectively, using the long-term exposure scenario. For the short-term exposure LOECs were 2 mg/L for both pesticides. For the reader measurement we could not observe any difference in the data of repeated measurements after thorough shaking, while the position of the embryo in the well turned out to be crucial for microscopic inspection. The assay allows to detect effects on zebrafish embryos at an early stage and at a low level of exposure clearly before deleterious consequences on an organ or organism level become visible. Since the embryos are kept alive, subsequent investigations of, e.g., EROD or micronucleus induction or further enzymatic biomarkers are possible. We will use the assay to investigate the impact of reactive toxicity on mechanism-specific biomarkers in zebrafish embryos. This would allow to verify whether biomarker results are representing a specific mode of action, or are rather the consequence of decreasing cell fitness.

22nd CIRP Conference on Life Cycle Engineering 2015 : Sydney, Australia, 7 - 9 April 2015 / ed.: Sami Kara 22nd CIRP Conference on Life Cycle Engineering, CIRP LCE 2015, Sidney, Australia, 7 Apr 2015 - 9 Apr 2015; Red Hook, NY : Curran, Procedia CIRP, 29, 609-614 (2015). doi:10.1016/j.procir.2015.02.206, Published by Curran, Red Hook, NY

Due to the multifaceted nature of sediments in combination with varying physical-chemical properties of environmental contaminants and a multitude of different living and feeding behaviours of the benthic community, estimation of the toxicological risk associated to polluted sediments appears to get easily hampered. Hence, for a maximum of veracity in sediment assessment, at least applied methods and technologies should provide reliability of gained results. However, little attention is brought to confounding factors that might influence the outcome of such applied research. Moreover, knowledge still lacks on how the various approaches compare and can be combined into a comprehensive investigation strategy. The present study aims at filling parts of these gaps through investigation and characterisation of (1) sediment extraction procedures and (2) whole sediment contact tests. After identifying the main confounding factors within preparation of exhaustive extracts, considerations are formulated regarding procedures with a reduced risk of altering samples. A novel vigorous but completely passive extraction method is then introduced, which works with dialysis over a semipermeable membrane (named membrane dialysis extraction, MDE). Initially, MDE extracts were compared to samples from the Soxhlet extraction protocol in biotests on cytotoxity, embryo toxicity and dioxin-like activity. Following a first application in a study on two riverine sediments, the new approach was part of a comprehensive comparison of five different vigorous leaching techniques. Extracts from another two riverine sediments were tested for their toxic effectiveness with respect to cytotoxity, embryo toxicity, and dioxin-like activity, and a number of compound classes, including PAHs, PCBs and DDXs, were determined chemically. Furthermore, three biomimetic extraction procedures were investigated in parallel. All techniques were evaluated in terms of extraction power and reproducibility. In a further comparative study, MDE, Soxhlet and two biomimetic methods were compared to direct contact exposure using the fish embryo test on Danio rerio. Summarizing all four studies, the ability of MDE, Soxhlet and ASE® to yield toxic substances from sediment samples was evaluated by ranking data from biotests and chemical analyses. The topic of sediment contact tests is initiated by an extensive literature review on the use of fish as a test organism in sediment investigation, covering also direct contact exposure. In order to allow reliable testing of sediments, six contact assays were thoroughly investigated for toxicity thresholds and test conditions. Subsequently, the contact test battery was applied on reference sediments spiked with a cocktail of either heavy metals or of organic substances. Resulting data were evaluated with respect to sensitivity and applicability of the different sediment contact tests. MDE was found to provide extraction powers comparable to automated ASE®-methods for most applications, while working at a reduced risk of alteration of resulting extracts. The Soxhlet procedure also gave overall good agreement with ASE®-based methods and MDE, but results indicate an elevated risk of loss of target analytes. Among the biomimetic approaches, Tenax proved best in replicating results from contact tests, while HBCD extracts revealed vigorous extraction powers in some cases. In general, biomimetic methods revealed strong variability of data among each other, complicating the investigation of the bioavailable contaminant fractions. With the sediment contact assays, toxicity thresholds were derived for all six investigated test systems. Furthermore, one natural as well as one formulated reference sediment for the whole test battery could be identified. The majority of assays returned clear dose-response relations for both sediments contaminated with either heavy metals of organic substances. However, the contact tests gave different sensitivities for the sediment-contaminant combinations and also showed varying steepness of the response curves. Based on the results and available literature data, sediment organic matter, clay content, substance properties and feeding as well as living habits were accounted for the observed differences in contaminant availability. The study concludes that investigations into sediment toxicity are likely to be influenced by a multitude of different parameters. As a consequence, it proposes a comprehensive strategical framework that involves several approaches in sediment toxicity assessment and points out required future developmental effort. Most pressing is an automated procedure for vigorous extraction at moderate temperatures, conceivable to be realized through a combination of ASE-methods and membrane dialysis. Furthermore, increased understanding of contaminant availability and test species behaviour is essential.

Environmental sciences Europe : ESEU 33(1), 106 (2021). doi:10.1186/s12302-021-00545-4, Published by Springer, Berlin ; Heidelberg

Environmental sciences Europe : ESEU 31, 44 (2019). doi:10.1186/s12302-019-0227-8, Published by Springer, Berlin

Environmental sciences Europe 31(1), 10 (2019). doi:10.1186/s12302-019-0192-2, Published by Springer21706, Heidelberg

Toxics 8(2), 45 (2020). doi:10.3390/toxics8020045, Published by MDPI, Basel