Search

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study was designed to verify the correlation between tinnitus and temporomandibular joint dysfunction.86 consecutive patients were enrolled in the study, all affected by subjective tinnitus without hearing impairment, from both genders, age between 18 and 60 years old. The final number of patients included in the study was 55. All patients received a temporo-mandibular joint examination. All the patients were asked to rate the severity of their symptoms before and after treatment using a VAS scale and the Tinnitus Handicap Inventory (THI) and they followed a standardized protocol for the investigation of tinnitus. All the subjects were monitored by the same researcher and they underwent the same splint treatment. The comparison between pre- and posttreatment phase scores showed in patients with predisposition of TMD and with TMD a statistically significant decrease of THI and VAS values. The characteristics of tinnitus and the degree of response to treatment confirmed the relationship between tinnitus and TMD. The authors believe that, when the most common causes of tinnitus, such as otologic disorders and neurological diseases are excluded, it is correct to evaluate the functionality of the temporo-mandibular joint and eventually treat its pathology to obtain tinnitus improvement or even resolution., (Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

The outcome of a five-year radiographic follow-up study of 150 patients with maxillo-mandibular malformations who had undergone Le Fort I osteotomy of the maxilla is reported. A superimposition technique made possible an exact evaluation of the adjustments effected. The results confirm the validity of a study conducted in 1977 and the five-year stability of the maxilla. The importance of the relationship between the Frankfort plane, occlusal plane and the osteotomy line is emphasized. In operations in which Le Fort I osteotomy of the maxilla is combined with a sagittal osteotomy of the mandible the maxilla undergoes minute displacements in the weeks following surgery, which can easily be predicted and allowed for at the planning stage. The long-term stability of the maxilla is assured.