Implementation of the CREED approach for environmental assessments.

Environmental exposure data are a key component of chemical and ecological assessments, supporting and guiding environmental management decisions and regulations. Measures taken to protect the environment based on exposure data can have social and economic implications. Flawed information may lead to measures being taken in the wrong place or to important action not being taken. Although the advantages of harmonizing evaluation methods have been demonstrated for hazard information, no comparable approach is established for exposure data evaluation. The goal of Criteria for Reporting and Evaluating Exposure Datasets (CREED) is to improve the transparency and consistency with which exposure data are evaluated regarding usability in environmental assessments. Here, we describe the synthesis of the CREED process, and propose methods and tools to summarize and interpret the outcomes of the data usability evaluation in support of decision-making and communication. The CREED outcome includes a summary that reports any key gaps or shortcomings in the reliability (data quality) and relevance (fitness for purpose) of the data being considered. The approach has been implemented in a workbook template (provided as Supporting Information), for assessors to readily follow the workflow and create a report card for any given dataset. The report card communicates the outcome of the CREED evaluation and summarizes important dataset attributes, providing a concise reference pertaining to the dataset usability for a specified purpose and documenting data limitations that may restrict data use or increase environmental assessment uncertainty. The application of CREED is demonstrated through three case studies, which also were used during beta testing of the methodology. As experience with the CREED approach application develops, further improvements may be identified and incorporated into the framework. Such development is to be encouraged in the interest of better science and decision-making, and to make environmental monitoring and assessment more cost-effective. Integr Environ Assess Manag 2024;00:1-16. © 2024 SETAC.

Clozapine modulation of zebrafish swimming behavior and gene expression as a case study to investigate effects of atypical drugs on aquatic organisms.

Mental illnesses affect more than 150 million people in Europe and lead to an increasing consumption of neuroactive drugs during the last twenty years. The antipsychotic compound, clozapine, is one of the most used psychotropic drugs worldwide, with potentially negative consequences for the aquatic environment. Hence, the objectives of the study presented here were the quantification of clozapine induced changes in swimming behavior of exposed Danio rerio embryos and the elucidation of the molecular effects on the serotonergic and dopaminergic systems. Yolk-sac larvae were exposed to different concentrations (0.2 mg/L, 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L and 6.4 mg/L) of clozapine for 116 h post-fertilization, and changes in the swimming behavior of the larvae were assessed. Further, quantitative real-time PCR was performed to analyze the expression of selected genes. The qualitative evaluation of changes in the swimming behavior of D. rerio larvae revealed a significant decrease of the average swimming distance and velocity in the light-dark transition test, with more than a 36% reduction at the highest exposure concentration of 6.4 mg/L. Furthermore, the total larval body length was reduced at the highest concentration. An in-depth analysis based on expression of selected target genes of the serotonin (slc6a4a) and dopamine (drd2a) system showed an upregulation at a concentration of 1.6 mg/L and above. In addition, a lower increase in expression was detected for biomarkers of general stress (adra1a and cyp1a2). Our data show that exposure to clozapine during development inhibits swimming activity of zebrafish larvae, which could, in part, be due to disruption of the serotonin- and dopamine system.

Minimum reporting standards based on a comprehensive review of the zebrafish embryo teratogenicity assay.

Reproductive toxicity chemical safety assessment involves extensive use of vertebrate animals for regulatory testing purposes. Although alternative methods such as the zebrafish embryo teratogenicity assay (identified in the present manuscript by the acronym ZETA) are promising for replacing tests with mammals, challenges to regulatory application involve lack of standardization and incomplete validation. To identify key protocol aspects and ultimately support improving this situation, a comprehensive review of the literature on the level of harmonization/standardization and validation status of the ZETA has been conducted. The gaps and needed advances of the available ZETA protocols were evaluated and discussed with respect to its applicability as an alternative approach for teratogenicity assessment. Based on the review outcomes, a set of minimum reporting standards for the experimental protocol is proposed. Together with other initiatives towards implementation of alternative approaches at the screening and regulatory levels, the application of minimum reporting requirements is anticipated to support future method standardization and validation, as well as identifying potential improvement aspects. Present findings are expected to ultimately support advancing the ongoing validation initiatives towards the regulatory acceptance of the ZETA.

Prior Antibiotic Therapy and the Onset of Healthcare-Associated Infections Sustained by Multidrug-Resistant Klebsiella pneumoniae in Intensive Care Unit Patients: A Nested Case-Control Study.

Epidemiological research has demonstrated direct relationships between antibiotic consumption and the emergence of multidrug-resistant (MDR) bacteria. In this nested case-control study, we assessed whether prior exposure to antibiotic therapy and its duration affect the onset of healthcare-associated infections (HAIs) sustained by MDR Klebsiella pneumoniae (MDR-Kp) in intensive care unit patients. Cases were defined as patients who developed an MDR-Kp HAI. Controls matched on sex and the length of intensive care unit (ICU) stay were randomly selected from the at-risk population. Any antibiotic agent received in systemic administration before the onset of infection was considered as antibiotic exposure. Multivariable conditional logistic regression analyses were performed to estimate the effect of prior exposure to each antibiotic class (Model 1) or its duration (Model 2) on the onset of HAIs sustained by MDR-Kp. Overall, 87 cases and 261 gender-matched controls were compared. In Model 1, aminoglycosides and linezolid independently increased the likelihood of developing an MDR-Kp HAI, whereas exposure to both linezolid and penicillins reduced the effect of linezolid alone. In Model 2, cumulative exposure to aminoglycosides increased the likelihood of the outcome, as well as cumulative exposures to penicillins and colistin, while a previous exposure to both penicillins and colistin reduced the influence of the two antibiotic classes alone. Our study confirms that aminoglycosides, penicillins, linezolid, and colistin may play a role in favoring the infections sustained by MDR-Kp. However, several double exposures in the time window before HAI onset seemed to hinder the selective pressure exerted by individual agents.

Application of screening tools for environmental hazard and risk to support assessment and subsequent prioritization of effluent discharges from the oil and gas industry.

Assessment and management of effluent discharges are key to avoiding environmental deterioration. Often compliance with discharge regulations and permits is based on a limited set of chemical parameters, while information on whole effluent hazardous properties (toxicity, bioaccumulation potential, persistence) and environmental risks is lacking. The need to collect those data and to become more effective in quickly identifying high-risk activities, without extensive laboratory testing, has led to the development of screening tools to complement information on chemical composition. A simple, Tier 1 screening "toolbox" is proposed which is comprised of solid-phase microextraction with gas chromatographic (SPME-GC) analysis, the in-vitro ecotoxicity assay Microtox, and a simple weathering assay. When combined with dilution modeling, screening-level risk assessments can be performed, providing additional lines of evidence to support a weight of evidence type of analysis. Application of the toolbox enables prioritization of discharges that may be deemed to require higher tier assessment. The toolbox was trialed on a number of produced water samples collected from offshore oil and gas facilities and effluents from petroleum processing and manufacturing sites. In contrast to what has been reported for petroleum products, results showed only moderate correlation between bioavailable hydrocarbons (bHCs) and toxicity, which might be related to the possible presence of toxic contaminants from other chemical classes or to methodological issues such as suboptimal conditions during transport. The methods employed were quick, inexpensive, and simple to conduct. They require relatively small volumes of sample, which is especially advantageous when evaluating discharges from remote offshore facilities. The toolbox adds valuable information on whole effluent properties to existing data, for example, on chemical composition, which can improve understanding of which discharges are more likely to pose a risk to the environment and so require further investigation or risk management. Integr Environ Assess Manag 2021;17:1025-1036. © 2021 Shell International B.V. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Towards elimination of measles and rubella in Italy: Progress and challenges.

INTRODUCTION: In the WHO European Region, endemic transmission of measles and rubella had been interrupted by 37 and 42 of the 53 member states (MSs), respectively, by 2018. Sixteen MSs are still endemic for measles, 11 for rubella and nine for both diseases, the latter including Italy. Elimination is documented by each country's National Verification Committee (NVC) through an annual status update (ASU). OBJECTIVE: By analysing data used to produce the ASUs, we aimed to describe the advances made by Italy towards elimination of measles and rubella. Moreover, we propose a set of major interventions that could facilitate the elimination process. METHODS: A total of 28 indicators were identified within the six core sections of the ASU form and these were evaluated for the period 2013-2018. These indicators relate to the incidence of measles/rubella; epidemiological investigation of cases; investigation of outbreaks; performance of the surveillance system; population immunity levels; and implementation of supplemental immunization activities (SIAs). RESULTS: From 2013 to 2018, epidemiological and laboratory analyses of measles cases in Italy improved substantially, allowing timely investigation in 2017 and 2018 of most outbreak and sporadic cases and identification of the majority of genotypic variants. Moreover, since 2017, vaccination coverage has increased significantly. Despite these improvements, several areas of concern emerged, prompting the following recommendations: i) improve outbreak monitoring; ii) strengthen the MoRoNet network; iii) increase the number of SIAs; iv) reinforce vaccination services; v) maintain regional monitoring; vi) design effective communication strategies; vii) foster the role of general practitioners and family paediatricians. CONCLUSIONS: The review of national ASUs is a crucial step to provide the NVC with useful insights into the elimination process and to guide the development of targeted interventions. Against this background, the seven recommendations proposed by the NVC have been shared with the Italian Ministry of Health and the Technical Advisory Group on measles and rubella elimination and have been incorporated into the new Italian Elimination Plan 2019-2023 as a technical aid to facilitate the achievement of disease elimination goals.

Optimization of screening-level risk assessment and priority selection of emerging pollutants - The case of pharmaceuticals in European surface waters.

Pharmaceuticals in surface waters have raised significant concern in recent years for their potential environmental effects. This study identified that at present a total of 477 substances (including 66 metabolites and transformation products) have been analyzed in European surface waters. Around 60% (284) of these compounds belonging to 16 different therapeutic groups were positively detected in one or more of 33 European countries. To conveniently and effectively prioritize potential high-risk compounds, an optimized method that considers the frequency of concentrations above predicted no effects levels was developed on the basis of the traditional method, and it was then used to identify and screen candidate priority pollutants in European surface waters. The results proved the feasibility and advantages of the optimized method. Pharmaceuticals detected in European surface waters were classified into 5 categories (high, moderate, endurable, negligible and safe) depending on their potential environmental effects and the distribution of pharmaceuticals. Circa 9% (45 out of 477) analyzed compounds showed a potential environmental risk to aquatic ecosystems. Among these 45 compounds, 12 compounds were indicated to have high environmental risk in aquatic environments, while 17 and 7 compounds showed moderate and small-scale environmental risks, respectively.

Behavioral profile alterations in zebrafish larvae exposed to environmentally relevant concentrations of eight priority pharmaceuticals.

Although the effects of pharmaceuticals on aquatic organisms have been widely investigated during the last decades, toxic effects, especially delayed toxicity, during the developmental stage at environmental relevant concentrations were rarely known. In this study, a sensitive assay based on behavioral alterations was used for studying the delayed toxicity during the developmental stage on zebrafish embryos. Eight pharmaceuticals that were frequently detected with concentrations ranging from ng/l to µg/l were screened for this study. Behavioral alterations of zebrafish at 118 hpf (hours post fertilization) after exposing to eight single pharmaceuticals with concentrations in the ranges of environmental detected and their mixtures during embryonic development (2-50 h post fertilization, hpf) were observed. Multiple endpoints, including mortality, hatching rate, swimming speed and angular velocity were evaluated. Results showed that behavioral profile alterations in zebrafish larvae are promising for predicting delayed sublethal effects of chemicals. Delayed hatch was observed at 72 hpf following embryonic exposure to triclosan (1 µg/l) and carbamazepine (100 µg/l) up to 50 hpf. The zebrafish larval locomotor behavior following embryonic exposure to 0.1 µg/l triclosan and 1 µg/l caffeine in the early stages of development (2-50 hpf) was altered. Furthermore, the effects of the mixture of 8 pharmaceuticals each with the highest environmental concentration on larval behavior were observed during embryonic development. Generally, this study showed that the effects of pharmaceuticals singly or their mixtures in surface waters cannot be ignored.

Integrated zebrafish-based tests as an investigation strategy for water quality assessment.

Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the "European Water Framework Directive" (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin-O-deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment.

p53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

Identification of Unknown Antiandrogenic Compounds in Surface Waters by Effect-Directed Analysis (EDA) Using a Parallel Fractionation Approach.

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 µg/L, equal to 71.4 µg flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure.

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters.

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

Diuron and diazinon alter the behavior of zebrafish embryos and larvae in the absence of acute toxicity.

The use of zebrafish for aquatic vertebrate (eco)toxicity testing allows the assessment of effects on a wide range of biological levels - from enzymes to sensory organs and behavioral endpoints. The present study investigated the effects of the insecticide diazinon and the herbicide diuron regarding the acute toxicity and behavior of zebrafish embryos and larvae. After conducting the fish embryo toxicity test, three concentrations (1, 2 and 3.5 mg L-1 for diazinon and 1, 2 and 3.8 mg L-1 for diuron) were evaluated for effects on embryonic spontaneous movement and heartbeat, larval light-dark transition response, and thigmotaxis. Although the modes-of-action are different, both pesticides proved to be moderately toxic to early life stages of zebrafish with 96 h LC50 of approximately 6.5 mg L-1 and similar EC50 values of approximately 4 mg L-1. Changes in behavioral endpoints were detected 24 h of exposure, suggesting that behavioral measurements can serve as sensitive and early indicators of pesticide exposure. Changes in behavior, such as decrease in spontaneous coiling movements of embryos and reduction of thigmotaxis in larvae, were pronounced for diuron, indicating the usefulness of the application of behavioral endpoints to assess the effects of other herbicides. In the case of diazinon, the effects were less prominent, but the detected changes in ratios between activity in light and darkness also point to the possibility of using behavioral changes for evaluation of insecticide effects. The obtained results support the usage of behavioral endpoints in zebrafish embryos and larvae for the detection of early effects of pesticides.

Assessment of a novel device for onsite integrative large-volume solid phase extraction of water samples to enable a comprehensive chemical and effect-based analysis.

The implementation of targeted and nontargeted chemical screening analysis in combination with in vitro and organism-level bioassays is a prerequisite for a more holistic monitoring of water quality in the future. For chemical analysis, little or no sample enrichment is often sufficient, while bioanalysis often requires larger sample volumes at a certain enrichment factor for conducting comprehensive bioassays on different endpoints or further effect-directed analysis (EDA). To avoid logistic and technical issues related to the storage and transport of large volumes of water, sampling would benefit greatly from onsite extraction. This study presents a novel onsite large volume solid phase extraction (LVSPE) device tailored to fulfill the requirements for the successful effect-based and chemical screening of water resources and complies with available international standards for automated sampling devices. Laboratory recovery experiments using 251 organic compounds in the log D range from -3.6 to 9.4 (at pH7.0) spiked into pristine water resulted in acceptable recoveries and from 60 to 123% for 159 out of 251 substances. Within a European-wide demonstration program, the LVSPE was able to enrich compounds in concentration ranges over three orders of magnitude (1ngL-1 to 2400ngL-1). It was possible to discriminate responsive samples from samples with no or only low effects in a set of six different bioassays (i.e. acetylcholinesterase and algal growth inhibition, androgenicity, estrogenicity, fish embryo toxicity, glucocorticoid activity). The LVSPE thus proved applicable for onsite extraction of sufficient amounts of water to investigate water quality thoroughly by means of chemical analysis and effect-based tools without the common limitations due to small sample volumes.

Bioassay battery interlaboratory investigation of emerging contaminants in spiked water extracts - Towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring.

Bioassays are particularly useful tools to link the chemical and ecological assessments in water quality monitoring. Different methods cover a broad range of toxicity mechanisms in diverse organisms, and account for risks posed by non-target compounds and mixtures. Many tests are already applied in chemical and waste assessments, and stakeholders from the science-police interface have recommended their integration in regulatory water quality monitoring. Still, there is a need to address bioassay suitability to evaluate water samples containing emerging pollutants, which are a current priority in water quality monitoring. The presented interlaboratory study (ILS) verified whether a battery of miniaturized bioassays, conducted in 11 different laboratories following their own protocols, would produce comparable results when applied to evaluate blinded samples consisting of a pristine water extract spiked with four emerging pollutants as single chemicals or mixtures, i.e. triclosan, acridine, 17α-ethinylestradiol (EE2) and 3-nitrobenzanthrone (3-NBA). Assays evaluated effects on aquatic organisms from three different trophic levels (algae, daphnids, zebrafish embryos) and mechanism-specific effects using in vitro estrogenicity (ER-Luc, YES) and mutagenicity (Ames fluctuation) assays. The test battery presented complementary sensitivity and specificity to evaluate the different blinded water extract spikes. Aquatic organisms differed in terms of sensitivity to triclosan (algae > daphnids > fish) and acridine (fish > daphnids > algae) spikes, confirming the complementary role of the three taxa for water quality assessment. Estrogenicity and mutagenicity assays identified with high precision the respective mechanism-specific effects of spikes even when non-specific toxicity occurred in mixture. For estrogenicity, although differences were observed between assays and models, EE2 spike relative induction EC50 values were comparable to the literature, and E2/EE2 equivalency factors reliably reflected the sample content. In the Ames, strong revertant induction occurred following 3-NBA spike incubation with the TA98 strain, which was of lower magnitude after metabolic transformation and when compared to TA100. Differences in experimental protocols, model organisms, and data analysis can be sources of variation, indicating that respective harmonized standard procedures should be followed when implementing bioassays in water monitoring. Together with other ongoing activities for the validation of a basic bioassay battery, the present study is an important step towards the implementation of bioanalytical monitoring tools in water quality assessment and monitoring.

Downscaling procedures reduce chemical use in androgen receptor reporter gene assay.

Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70µL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250µL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200µL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC50 (3.4-3.7×10(-10)M) and flutamide IC50 (2.2-3.3×10(-7)M) values very similar to those from regular assays (3.1-4.2×10(-10) and 2.1-3.3×10(-7)M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC50 values for 1-hydroxypyrene (2.1-2.8×10(-6)M), bisphenol A (2.6-3.3×10(-6)M), and triclosan (1.2-1.9×10(-6)M) in agreement with values obtained through high-volume exposure (2.3-2.8, 2.5-3.4 and 1.0-1.3×10(-6)M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays.

Effect-directed analysis supporting monitoring of aquatic environments--An in-depth overview.

Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.

Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae.

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3-3000ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300 ng/L and 3000 ng/L) as well as growth impairment and delayed development (100 ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169 ng/L) was similar to the LC50 value at 8 dpf (188 and 202 ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species.

Black carbon inclusive multichemical modeling of PBDE and PCB biomagnification and -transformation in estuarine food webs.

Bioavailability and bioaccumulation of polybrominated diphenylethers (PBDEs) are affected by adsorption on black carbon (BC) and metabolism in biota, respectively. Recent studies have addressed these two processes separately, illustrating their importance in assessing contaminant dynamics. In order to properly examine biomagnification of polychlorinated biphenyls (PCBs) and PBDEs in an estuarine food-web, here we set up a black carbon inclusive multichemical model. A dual domain sorption model, which accounted for sorption to organic matter (OM) and black carbon (BC), was used to estimate aqueous phase concentrations from the measured chemical concentrations in suspended solids. We adapted a previously published multichemical model that tracks the movement of a parent compound and its metabolites in each organism and within its food web. First, the model was calibrated for seven PCB congeners assuming negligible metabolism. Subsequently, PBDE biomagnification was modeled, including biotransformation and bioformation of PBDE congeners, keeping the other model parameters the same. The integrated model was capable of predicting trophic magnification factors (TMF) within error limits. PBDE metabolic half-lives ranged 21-415 days and agreed to literature data. The results showed importance of including BC as an adsorbing phase, and biotransformation and bioformation of PBDEs for a proper assessment of their dynamics in aquatic systems.