Biochemistry of microwave controlled Heracleum sosnowskyi (Manden.) roots with an ecotoxicological aspect.

Sosnowski hogweed is an invasive weed in eastern-middle Europe that is dangerous to human health and the environment. The efficacy of its control using chemical and mechanical methods is limited. Electromagnetic radiation (microwaves) could be an environmentally friendly alternative for controlling this species. This study aims to: (1) Determine the effect of varying microwave treatment (MWT) durations on the control of S. hogweed using a device emitting microwaves at 2.45 GHz, 32.8 kW/m2; (2) Evaluate the impact of MWT on soil by an ecotoxicological bioassays; (3) Analyze biochemical changes occurring in the roots during the process. A field study was performed to assess the efficacy of S. hogweed control using MWT in times from 2.5 to 15 min. The MWT-treated soil was collected immediately after treatment (AT) and tested using bioassays (Phytotoxkit, Ostracodtoxkit, and Microtox). Fourteen days AT, the MWT hogweed roots were dug out, air-dried, and analyzed for the content and composition of essential oil, sugars, and fatty acids. According to the ecotoxicological biotests, the MWT soils were classified as non-toxic or low-toxic. The regeneration of hogweed was observed only in non-treated plants (control). Hogweed MWT for 2.5-15 min did not regenerate up to 14 days AT. The average weight of roots in hogweed MWT for 15.0 min was ca. two times smaller than the control plants. Those roots contained significantly higher amounts of sugars and saturated fatty acids than the control. We did not find a correlation between S. hogweed root essential oil content and composition and MWT time. The main compounds of essential oil were p­cymene and myristicin. No highly photosensitizing compounds were identified in the tested root oil. We conclude that MWT of S. hogweed could be an environmentally safe and prospective control method, but more studies are needed.

Ecotoxicological effects of tungsten on celery (Apium graveolens L) and pepper (Capsicum spp.).

Background: Tungsten (W) is an emerging heavy metal pollutant, yet research remains scarce on the biomonitor and sensitive biomarkers for W contamination. Methods: In this study, celery and pepper were chosen as study subjects and subjected to exposure cultivation in solutions with five different levels of W. The physiological and biochemical toxicities of W on these two plants were systematically analyzed. The feasibility of utilizing celery and pepper as biomonitor organisms for W contamination was explored and indicative biomarkers were screened. Results: The results indicated that W could inhibit plants' root length, shoot height, and fresh weight while concurrently promoting membrane lipid peroxidation. Additionally, W enhanced the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and total antioxidant capacity (TAOC) to counteract oxidative damage. From a physiological perspective, pepper exhibited potential as a biomonitor for W contamination. Biochemical indicators suggested that SOD could serve as a sensitive biomarker for W in celery, while TAOC and POD were more suitable for the roots and leaves of pepper. In conclusion, our study investigated the toxic effects of W on celery and pepper, contributing to the understanding of W's environmental toxicity. Furthermore, it provided insights for selecting biomonitor organisms and sensitive biomarkers for W contamination.

Reviewing the use of zebrafish for the detection of neurotoxicity induced by chemical mixtures through the analysis of behaviour.

The knowledge and assessment of mixtures of chemical pollutants in the aquatic environment is a complex issue that is often challenging to address. In this review, we focused on the use of zebrafish (Danio rerio), a vertebrate widely used in biomedical research, as a model for detecting the effects of chemical mixtures with a focus on behaviour. Our aim was to summarize the current status of the ecotoxicological research in this sector. Specifically, we limited our research to the period between January 2012 and September 2023, including only those works aimed at detecting neurotoxicity through behavioural endpoints, utilizing zebrafish at one or more developmental stages, from egg to adult. Additionally, we gathered the findings for every group of chemicals involved and summarised data from all the works we included. At the end of the screening process 101 papers were considered eligible for inclusion. Results show a growing interest in zebrafish at all life stages for this kind of research in the last decade. Also, a wide variety of different assays, involving different senses, was used in the works we surveyed, with exposures ranging from acute to chronic. In conclusion, the results of this study show the versatility of zebrafish as a model for the detection of mixture toxicity although, for what concerns behavioural analysis, the lack of standardisation of methods and endpoints might still be limiting.

Lipid metabolism in crocodilians: A field with promising applications in the field of ecotoxicology.

In the last years, lipid physiology has become an important research target for systems biology applied to the field of ecotoxicology. Lipids are not only essential components of biological membranes, but also participate in extra and intracellular signaling processes and as signal transducers and amplifiers of regulatory cascades. Particularly in sauropsids, lipids are the main source of energy for reproduction, growth, and embryonic development. In nature, organisms are exposed to different stressors, such as parasites, diseases and environmental contaminants, which interact with lipid signaling and metabolic pathways, disrupting lipid homeostasis. The system biology approach applied to ecotoxicological studies is crucial to evaluate metabolic regulation under environmental stress produced by xenobiotics. In this review, we cover information of molecular mechanisms that contribute to lipid metabolism homeostasis in sauropsids, specifically in crocodilian species. We focus on the role of lipid metabolism as a powerful source of energy and its importance during oocyte maturation, which has been increasingly recognized in many species, but information is still scarce in crocodiles. Finally, we highlight priorities for future research on the influence of environmental stressors on lipid metabolism, their potential effect on the reproductive system and thus on the offspring, and their implications on crocodilians conservation.

Ecotoxicological assessments of atmospheric biomonitors exposed to urban pollution in a Brazilian metropolis.

Subcellular metal distribution assessments are the most adequate biomonitoring approach to evaluate metal toxicity, instead of total metal assessments This study aimed to assess subcellular metal distributions and associations to the main metal exposure biomarker, metallothionein (MT), in two bromeliad species (Tillandsia usneoides and Tillandsia stricta) exposed established in industrial, urban, and port areas in the metropolitan region of Rio de Janeiro, southeastern Brazil, through an active biomonitoring approach conducted one year. Metals and metalloids in three subcellular fractions (insoluble, thermolabile and thermostable) obtained from the MT purification process were determined by inductively coupled plasma mass spectrometry (ICP-MS). Lower MT concentrations were observed both during the dry sampling periods, associated to the crassulacean acid metabolism (CAM) and during the COVID-19 pandemic, due to reduced urban mobility, decreasing pollutant emissions. The percentage of non-bioavailable metals detected in the insoluble fraction increased throughout the sampling period for both species. Several metals (Cr, Co, Cu, Cd, Mn, Ni, Se, and Zn), most associated with vehicle emissions, the main pollutant source in urban centers, were detected in the thermostable fraction and are, thus, associated with MT through the MT-metal detoxification route. Insoluble metal concentrations were higher in T. stricta, indicating that this species seems less susceptible to cellular metal exposure damage. A potential protective effect of Se and Fe was detected against Pb, suggested by a strong negative correlation, which may be attributed to antioxidant roles and similar uptake routes, respectively.

Development of a rapid screening method utilizing 2D LC for effect-directed analysis in the identification of environmental toxicants.

Effect-directed analysis (EDA) is a crucial tool in environmental toxicology, effectively integrating toxicity testing with chemical analysis. The conventional EDA approach, however, presents challenges such as significant solvent consumption, extended analysis time, labor intensity, and potential contamination risks. In response, we introduce an innovative alternative to the conventional EDA. This method utilizes the MTT bioassay and online two-dimensional liquid chromatography (2D LC) coupled with high-resolution mass spectrometry (HR-MS), significantly reducing the fractionation steps and leveraging the enhanced sensitivity of the bioassay and automated chemical analysis. In the chemical analysis phase, a switching valve interface is employed for comprehensive analysis. We tested the performance of both the conventional and our online 2D LC-based methods using a household product. Both methods identified the same number of toxicants in the sample. Our alternative EDA is 22.5 times faster than the conventional method, fully automated, and substantially reduces solvent consumption. This novel approach offers ease, cost-effectiveness, and represents a paradigm shift in EDA methodologies. By integrating a sensitive bioassay with online 2D LC, it not only enhances efficiency but also addresses the challenges associated with traditional methods, marking a significant advancement in environmental toxicology research.

Bio-QSARs 2.0: Unlocking a new level of predictive power for machine learning-based ecotoxicity predictions by exploiting chemical and biological information.

Practical, legal, and ethical reasons necessitate the development of methods to replace animal experiments. Computational techniques to acquire information that traditionally relied on animal testing are considered a crucial pillar among these so-called new approach methodologies. In this light, we recently introduced the Bio-QSAR concept for multispecies aquatic toxicity regression tasks. These machine learning models, trained on both chemical and biological information, are capable of both cross-chemical and cross-species predictions. Here, we significantly extend these models' applicability. This was realized by increasing the quantity of training data by a factor of approximately 20, accomplished by considering both additional chemicals and aquatic organisms. Additionally, variable test durations and associated random effects were accommodated by employing a machine learning algorithm that combines tree-boosting with mixed-effects modeling (i.e., Gaussian Process Boosting). We also explored various biological descriptors including Dynamic Energy Budget model parameters, taxonomic distances, as well as genus-specific traits and investigated the inclusion of mode-of-action information. Through these efforts, we developed Bio-QSARs for fish and aquatic invertebrates with exceptional predictive power (R squared of up to 0.92 on independent test sets). Moreover, we made considerable strides to make models applicable for a range of use cases in environmental risk assessment as well as research and development of chemicals. Models were made fully explainable by implementing an algorithmic multicollinearity correction combined with SHapley Additive exPlanations. Furthermore, we devised novel approaches for applicability domain construction that take feature importance into account. We are hence confident these models, which are available via open access, will make a significant contribution towards the implementation of new approach methodologies and ultimately have the potential to support "Green Chemistry" and "Green Toxicology".

An introduction to Criteria for Reporting and Evaluating Exposure Datasets (CREED) for use in environmental assessments.

Risks posed by environmental exposure to chemicals are routinely assessed to inform activities ranging from environmental status reporting to authorization and registration of chemicals for commercial uses. Environmental risk assessment generally relies on two key values generated from exposure data and ecotoxicity data. Data sets of measured concentrations of chemicals in environmental matrices, referred to here as exposure data, are widely used to support environmental risk management, decision-making, and reporting, such as for chemical screening, ecological or human health risk assessments, and establishment of guidelines. Practitioners have developed schemes to determine the suitability of ecotoxicity data for specific purposes, focused on evaluating reliability and relevance, but analogous schemes are not available for exposure data. Moreover, regulatory guidance arguably provides less resolution on reporting and evaluating exposure data sets compared to ecotoxicity data. The evaluation of exposure data sets is subject to limitations from variable or unreported data quality objectives and/or from differences in expert judgments, potentially introducing bias and leading to decisions based on flawed and/or inconsistent information. Exposure data sets should be evaluated for reliability and relevance prior to use in environmental assessments. This paper is the first of a four-paper series detailing the outcomes of a Society of Environmental Toxicology and Chemistry technical workshop that has developed Criteria for Reporting and Evaluating Exposure Datasets (CREED). The workshop participants developed practical, systematic criteria for consistent and transparent evaluation of the reliability (quality) and relevance (fitness for purpose) of exposure data. This guidance should apply to many different (unspecified) purposes of assessment. CREED can be used to evaluate existing data sets, but can also inform data generators interested in improving their data collection and reporting to maximize data utility to other users. This first paper details existing frameworks for the evaluation of exposure data sets and demonstrates the need for CREED, drawing from different regulatory assessments, and describes the technical workshop. Integr Environ Assess Manag 2024;20:975-980. © 2024 SETAC.

Identifying and Predicting Delayed Mortality with Toxicokinetic-Toxicodynamic Models.

The prevalence of standardized toxicity testing in ecotoxicology has largely obscured the notion that toxicity is a function of time as well. The necessity of considering time is vividly demonstrated by observations of delayed mortality, that is, deaths continue to occur even when animals are no longer exposed to a toxicant. In this contribution, I explore to what extent toxicokinetic-toxicodynamic (TKTD) models from the framework of the General Unified Threshold model for Survival (GUTS) can capture delayed mortality, and to what extent this phenomenon can be predicted from short-term standard tests. I use a previously published data set for fluoroquinolones in Daphnia magna that shows strongly delayed mortality (using immobilization as a proxy for death). The model analysis shows that the GUTS stochastic death models can capture delayed mortality in the complete data set with a long recovery phase, but that the delayed effects would not have been predicted from a 2-day standard test. The study underlines the limited information content of standard acute test designs. Toxicokinetic-toxicodynamic modeling offers a handle on the time aspects of toxicity but cannot always be relied on to provide accurate extrapolations based on severely limited standard tests. The phenomenon of delayed toxicity requires more structured study to clarify its prevalence and impact; I discuss several avenues for further investigation. Environ Toxicol Chem 2024;43:1030-1035. © 2024 SETAC.

Ecotoxicological assessment of biomass-derived furan platform chemicals using aquatic and terrestrial bioassays.

An environmental toxicological assessment of fourteen furanic compounds serving as valuable building blocks produced from biomass was performed. The molecules selected included well studied compounds serving as control examples to compare the toxicity exerted against a variety of highly novel furans which have been additionally targeted as potential or current alternatives to biofuels, building blocks and polymer monomers. The impact of the furan platform chemicals targeted on widely applied ecotoxicity model organisms was determined employing the marine bioluminescent bacterium Aliivibrio fischeri and the freshwater green microalgae Raphidocelis subcapitata, while their ecotoxicity effects on plants were assessed using dicotyledonous plants Sinapis alba and Lepidium sativum. Regarding the specific endpoints evaluated, the furans tested were slightly toxic or practically nontoxic for A. fischeri following 5 and 15 min of exposure. Moreover, most of the building blocks did not affect the growth of L. sativum and S. alba at 150 mg L-1 for 72 h of exposure. Specifically, 9 and 11 out of the 14 furan platform chemicals tested were non-effective or stimulant for L. sativum and S. alba respectively. Given that furans comprise common inhibitors in biorefinery fermentations, the growth inhibition of the specific building blocks was studied using the industrial workhorse yeast Saccharomyces cerevisiae, demonstrating insignificant inhibition on eukaryotic cell growth following 6, 12 and 16 h of exposure at a concentration of 500 mg L-1. The study provides baseline information to unravel the ecotoxic effects and to confirm the green aspects of a range of versatile biobased platform molecules.

Big Question to Developing Solutions: A Decade of Progress in the Development of Aquatic New Approach Methodologies from 2012 to 2022.

In 2012, 20 key questions related to hazard and exposure assessment and environmental and health risks of pharmaceuticals and personal care products in the natural environment were identified. A decade later, this article examines the current level of knowledge around one of the lowest-ranking questions at that time, number 19: "Can nonanimal testing methods be developed that will provide equivalent or better hazard data compared with current in vivo methods?" The inclusion of alternative methods that replace, reduce, or refine animal testing within the regulatory context of risk and hazard assessment of chemicals generally faces many hurdles, although this varies both by organism (human-centric vs. other), sector, and geographical region or country. Focusing on the past 10 years, only works that might reasonably be considered to contribute to advancements in the field of aquatic environmental risk assessment are highlighted. Particular attention is paid to methods of contemporary interest and importance, representing progress in (1) the development of methods which provide equivalent or better data compared with current in vivo methods such as bioaccumulation, (2) weight of evidence, or (3) -omic-based applications. Evolution and convergence of these risk assessment areas offer the basis for fundamental frameshifts in how data are collated and used for the protection of taxa across the breadth of the aquatic environment. Looking to the future, we are at a tipping point, with a need for a global and inclusive approach to establish consensus. Bringing together these methods (both new and old) for regulatory assessment and decision-making will require a concerted effort and orchestration. Environ Toxicol Chem 2024;43:559-574. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Methods for estimating no-effect toxicity concentrations in ecotoxicology.

A range of new statistical approaches is being developed and/or adopted in ecotoxicology that, when combined, can greatly improve the estimation of no-effect toxicity values from concentration-response (CR) experimental data. In particular, we compare the existing no-effect-concentration (NEC) threshold-based toxicity metric with an alternative no-significant-effect-concentration (NSEC) metric suitable for when CR data do not show evidence of a threshold effect. Using a model-averaging approach, these metrics can be combined to yield estimates of N(S)EC and of their uncertainty within a single analysis framework. The outcome is a framework for CR analysis that is robust to uncertainty in the model formulation, and for which resulting estimates can be confidently integrated into risk assessment frameworks, such as the species sensitivity distribution (SSD). Integr Environ Assess Manag 2024;20:279-293. © 2023 Commonwealth of Australia and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Cross-Species Extrapolation of Biological Data to Guide the Environmental Safety Assessment of Pharmaceuticals-The State of the Art and Future Priorities.

The extrapolation of biological data across species is a key aspect of biomedical research and drug development. In this context, comparative biology considerations are applied with the goal of understanding human disease and guiding the development of effective and safe medicines. However, the widespread occurrence of pharmaceuticals in the environment and the need to assess the risk posed to wildlife have prompted a renewed interest in the extrapolation of pharmacological and toxicological data across the entire tree of life. To address this challenge, a biological "read-across" approach, based on the use of mammalian data to inform toxicity predictions in wildlife species, has been proposed as an effective way to streamline the environmental safety assessment of pharmaceuticals. Yet, how effective has this approach been, and are we any closer to being able to accurately predict environmental risk based on known human risk? We discuss the main theoretical and experimental advancements achieved in the last 10 years of research in this field. We propose that a better understanding of the functional conservation of drug targets across species and of the quantitative relationship between target modulation and adverse effects should be considered as future research priorities. This pharmacodynamic focus should be complemented with the application of higher-throughput experimental and computational approaches to accelerate the prediction of internal exposure dynamics. The translation of comparative (eco)toxicology research into real-world applications, however, relies on the (limited) availability of experts with the skill set needed to navigate the complexity of the problem; hence, we also call for synergistic multistakeholder efforts to support and strengthen comparative toxicology research and education at a global level. Environ Toxicol Chem 2024;43:513-525. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Editorial trend: adverse outcome pathway (AOP) and computational strategy - towards new perspectives in ecotoxicology.

The adverse outcome pathway (AOP) has been conceptualized in 2010 as an analytical construct to describe a sequential chain of causal links between key events, from a molecular initiating event leading to an adverse outcome (AO), considering several levels of biological organization. An AOP aims to identify and organize available knowledge about toxic effects of chemicals and drugs, either in ecotoxicology or toxicology, and it can be helpful in both basic and applied research and serve as a decision-making tool in support of regulatory risk assessment. The AOP concept has evolved since its introduction, and recent research in toxicology, based on integrative systems biology and artificial intelligence, gave it a new dimension. This innovative in silico strategy can help to decipher mechanisms of action and AOP and offers new perspectives in AOP development. However, to date, this strategy has not yet been applied to ecotoxicology. In this context, the main objective of this short article is to discuss the relevance and feasibility of transferring this strategy to ecotoxicology. One of the challenges to be discussed is the level of organisation that is relevant to address for the AO (population/community). This strategy also offers many advantages that could be fruitful in ecotoxicology and overcome the lack of time, such as the rapid identification of data available at a time t, or the identification of "data gaps". Finally, this article proposes a step forward with suggested priority topics in ecotoxicology that could benefit from this strategy.

Second Fundão Dam Rupture Science Meeting: Updating the science.

The second Fundão Dam Rupture Science Meeting was held in Ouro Preto, Minas Gerais, Brazil, on 21 and 22 September 2022. A total of 100 delegates attended the meeting, which featured high-quality oral presentations and posters over the two days and lively discussions of the science presented. The meeting resulted in nine papers being published in this special series. In the discussion, it was commented that the first meeting had been more a meeting of competitive perspectives but that the second meeting featured a more collaborative sharing of knowledge. Also, there had been substantial improvement in ways to deal with the lack of pre-event data and account for other stressors in the system, and as a result, stronger conclusions regarding the recovery rates of the system were able to be made. Importantly, there was no evidence for a feasible "time bomb" of a future catastrophic release of contaminants, but it was agreed that different scenarios continue to be researched. Integr Environ Assess Manag 2024;20:70-73. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Reporting and reproducibility: Proteomics of fish models in environmental toxicology and ecotoxicology.

Environmental toxicology and ecotoxicology research efforts are employing proteomics with fish models as New Approach Methodologies, along with in silico, in vitro and other omics techniques to elucidate hazards of toxicants and toxins. We performed a critical review of toxicology studies with fish models using proteomics and reported fundamental parameters across experimental design, sample preparation, mass spectrometry, and bioinformatics of fish, which represent alternative vertebrate models in environmental toxicology, and routinely studied animals in ecotoxicology. We observed inconsistencies in reporting and methodologies among experimental designs, sample preparations, data acquisitions and bioinformatics, which can affect reproducibility of experimental results. We identified a distinct need to develop reporting guidelines for proteomics use in environmental toxicology and ecotoxicology, increased QA/QC throughout studies, and method optimization with an emphasis on reducing inconsistencies among studies. Several recommendations are offered as logical steps to advance development and application of this emerging research area to understand chemical hazards to public health and the environment.

Cryptic Species in Ecotoxicology.

The advent of genetic methods has led to the discovery of an increasing number of species that previously could not be distinguished from each other on the basis of morphological characteristics. Even though there has been an exponential growth of publications on cryptic species, such species are rarely considered in ecotoxicology. Thus, the particular question of ecological differentiation and the sensitivity of closely related cryptic species is rarely addressed. Tackling this question, however, is of key importance for evolutionary ecology, conservation biology, and, in particular, regulatory ecotoxicology. At the same time, the use of species with (known or unknown) cryptic diversity might be a reason for the lack of reproducibility of ecotoxicological experiments and implies a false extrapolation of the findings. Our critical review includes a database and literature search through which we investigated how many of the species most frequently used in ecotoxicological assessments show evidence of cryptic diversity. We found a high proportion of reports indicating overlooked species diversity, especially in invertebrates. In terrestrial and aquatic realms, at least 67% and 54% of commonly used species, respectively, were identified as cryptic species complexes. The issue is less prominent in vertebrates, in which we found evidence for cryptic species complexes in 27% of aquatic and 6.7% of terrestrial vertebrates. We further exemplified why different evolutionary histories may significantly determine cryptic species' ecology and sensitivity to pollutants. This in turn may have a major impact on the results of ecotoxicological tests and, consequently, the outcome of environmental risk assessments. Finally, we provide a brief guideline on how to deal practically with cryptic diversity in ecotoxicological studies in general and its implementation in risk assessment procedures in particular. Environ Toxicol Chem 2023;42:1889-1914. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Antidepressant pharmaceuticals in aquatic systems, individual-level ecotoxicological effects: growth, survival and behavior.

The growing consumption of antidepressant pharmaceuticals has resulted in their widespread occurrence in the environment, particularly in waterways with a typical concentration range from ng L-1 to µg L-1. An increasing number of studies have confirmed the ecotoxic potency of antidepressants, not only at high concentrations but also at environmentally relevant levels. The present review covers literature from the last decade on the individual-level ecotoxicological effects of the most commonly used antidepressants, including their impact on behavior, growth, and survival. We focus on the relationship between antidepressants physico-chemical properties and dynamics in the environment. Furthermore, we discuss the advantages of considering behavioral changes as sensitive endpoints in ecotoxicology, as well as some current methodological shortcomings in the field, including low standardization, reproducibility and context-dependency.

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

Chemical Mixtures and Multiple Stressors: Same but Different?

Ecosystems are strongly influenced by multiple anthropogenic stressors, including a wide range of chemicals and their mixtures. Studies on the effects of multiple stressors have largely focussed on nonchemical stressors, whereas studies on chemical mixtures have largely ignored other stressors. However, both research areas face similar challenges and require similar tools and methods to predict the joint effects of chemicals or nonchemical stressors, and frameworks to integrate multiple chemical and nonchemical stressors are missing. We provide an overview of the research paradigms, tools, and methods commonly used in multiple stressor and chemical mixture research and discuss potential domains of cross-fertilization and joint challenges. First, we compare the general paradigms of ecotoxicology and (applied) ecology to explain the historical divide. Subsequently, we compare methods and approaches for the identification of interactions, stressor characterization, and designing experiments. We suggest that both multiple stressor and chemical mixture research are too focused on interactions and would benefit from integration regarding null model selection. Stressor characterization is typically more costly for chemical mixtures. While for chemical mixtures comprehensive classification systems at suborganismal level have been developed, recent classification systems for multiple stressors account for environmental context. Both research areas suffer from rather simplified experimental designs that focus on only a limited number of stressors, chemicals, and treatments. We discuss concepts that can guide more realistic designs capturing spatiotemporal stressor dynamics. We suggest that process-based and data-driven models are particularly promising to tackle the challenge of prediction of effects of chemical mixtures and nonchemical stressors on (meta-)communities and (meta-)food webs. We propose a framework to integrate the assessment of effects for multiple stressors and chemical mixtures. Environ Toxicol Chem 2023;42:1915-1936. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.