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The validation of high-throughput toxicity tests with invertebrate species is a key priority to improve hazard assessment of new chemicals and increase the available test guidelines with organisms from a representative set of taxa. This work aimed to contribute to the validation of an embryo test with the freshwater gastropod Lymnaea stagnalis, which has been identified by Organization for Economic Co-operation and Development (OECD) as a potential invertebrate test model, and provide the basis for such an endeavor. Recently, a L. stagnalis reproductive test was standardized by the OECD. However, to encompass the entire life cycle, it is crucial to addresses embryogenic development - a phase highly susceptible to various anthropogenic chemicals, which is covered in the proposed methodology. The approach used in the present study is in line with the OECD guidelines and other published studies, namely the Detailed Review Paper (DRP) on Mollusks life-cycle toxicity testing. Here, the assay quality criteria such as basal mortality and abnormality rates, development, growth and hatching rates, the appropriated testing media, and the optimal assay duration were investigated. Cadmium was chosen as the positive test substance, due to the available data and the verified model sensitivity to this compound, namely in the OECD reproductive test validation process. The obtained data demonstrate that L. stagnalis embryogenesis using the developed methodology is highly sensitive to cadmium. High concentration-response correlation was observed using this reference compound, the EC10 and EC50 for growth are 13.57 and 21.84 μg/L, respectively, after 168 h of exposure. The development EC's 10 and 50 were 15.75 and 38.66 μg/L, respectively, after 240 h. This demonstrates the model sensitivity to this compound when compared with other embryo test models, as well as the model sensitivity during the embryogenesis, if compared with the adult stage. Further, given the determined sensitivity parameters, and incubation times, the test can be performed at 240 h as over 95 % of the control embryos were hatched and no further significant changes in the exposure groups were determined. Overall, the findings of the present study demonstrate that the embryo test with L. stagnalis has potential to high-throughput testing and the model has a high sensitivity to cadmium during this life cycle period. The background data provide by this study will be essential to foster the future standardization of this assay., 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 © 2023 Elsevier B.V. All rights reserved.)

Recent reports raise the concern that exposure to several environmental chemicals may induce persistent changes that go beyond the exposed organisms, being transferred to subsequent generations even in the absence of the original chemical insult. These changes in subsequent non-exposed generations have been related to epigenetic changes. Although highly relevant for hazard and risk assessment, biomarkers of epigenetic modifications that can be associated with adversity, are still not integrated into hazard assessment frameworks. Here, in order to validate new biomarkers of epigenetic modifications in a popular animal model, zebrafish embryos were exposed to different concentrations of Bisphenol A (0.01, 0.1, 1 and 10 mg/L) and Valproic Acid (0.8, 4, 20 and 100 mg/L), two chemicals reported to alter the modulation of the epigenome. Morphological abnormalities and epigenetic changes were assessed at 80 hours-post fertilization, including DNA global methylation and gene expression of both DNA and histone epigenetic modifications. Gene expression changes were detected at concentrations below those inducing morphological abnormalities. These results further support the importance of combining epigenetic biomarkers with apical endpoints to improve guidelines for chemical testing and hazard assessment, and favour the integration of new biomarkers of epigenetic modifications into the standardized OECD test guideline 236 with zebrafish embryos., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This review article gathers the available information on the use of embryo-tests as high-throughput tools for toxicity screening, hazard assessment and prioritization of new and existing chemical compounds. The approach is contextualized considering the new legal trends for animal experimentation, fostering the 3R policy, with reduction of experimental animals, addressing the potential of embryo-tests as high-throughput toxicity screening and prioritizing tools. Further, the current test guidelines, such as the ones provided by OECD and EPA, focus mainly in a limited number of animal lineages, particularly vertebrates and arthropods. To extrapolate hazard assessment to the ecosystem scale, a larger diversity of taxa should be tested. The use of new experimental animal models in toxicity testing, from a representative set of taxa, was thoroughly revised and discussed in this review. Here, we critically review current tools and the main advantages and drawbacks of different animal models and set researcher priorities., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Obesity, a risk factor for the development of type-2 diabetes, hypertension, cardiovascular disease, hepatic steatosis and some cancers, has been ranked in the top 10 health risk in the world by the World Health Organization. Despite the growing body of literature evidencing an association between the obesity epidemic and specific chemical exposure across a wide range of animal taxa, very few studies assessed the effects of chemical mixtures and environmental samples on lipid homeostasis. Additionally, the mode of action of several chemicals reported to alter lipid homeostasis is still poorly understood. Aiming to fill some of these gaps, we combined an in vivo assay with the model species zebrafish (Danio rerio) to screen lipid accumulation and evaluate expression changes of key genes involved in lipid homeostasis, alongside with an in vitro transactivation assay using human and zebrafish nuclear receptors, retinoid X receptor α and peroxisome proliferator-activated receptor γ. Zebrafish larvae were exposed from 4 th day post-fertilization until the end of the experiment (day 18), to six different treatments: experimental control, solvent control, tributyltin at 100 ng/L Sn and 200 ng/L Sn (positive control), and wastewater treatment plant influent at 1.25% and 2.5%. Exposure to tributyltin and to 2.5% influent led to a significant accumulation of lipids, with white adipose tissue deposits concentrating in the perivisceral area. The highest in vitro tested influent concentration (10%) was able to significantly transactivate the human heterodimer PPARγ/RXRα, thus suggesting the presence in the influent of HsPPARγ/RXRα agonists. Our results demonstrate, for the first time, the ability of complex environmental samples from a municipal waste water treatment plant influent to induce lipid accumulation in zebrafish larvae., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Literature data and data obtained with modelling tools were compiled to derive the physicochemical behaviour of 24 priority Hazardous and Noxious Substances (HNS), as a proxy to improve environmental, public health and political issues in relation to HNS spills. Parameters that rule the HNS behaviour in water and those that determine their distribution and persistence in the environment, such as fugacity, physicochemical degradation, biodegradation, bioaccumulation/biotransformation and aquatic toxicity, were selected. Data systematized and produced in the frame of the Arcopol Platform project was made available through a public database (http://www.ciimar.up.pt/hns/substances.php). This tool is expected to assist stakeholders involved in HNS spills preparedness and response, policy makers and legislators, as well as to contribute to a current picture of the scientific knowledge on the fate, behaviour, weathering and toxicity of priority HNS, being essential to support future improvements in maritime safety and coastal pollution response before, during and after spill incidents., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fish are capable of sensing water-borne chemicals at sub-lethal concentrations. Inadequate behavioral responses to physiological and environmental stimuli owing to adverse effects of aquatic toxicants can have serious implications for survival. In this study we exposed juvenile rainbow trout (Oncorhynchus mykiss) during 5 days to a low-concentration mixture of three co-occurring herbicides: atrazine, linuron and metolachlor, at maximum concentrations of 4.5, 4.9 and 13.4 μg L(-1), respectively. Our hypothesis was that fish behavior - swimming activity and interactions between individuals - would be modified due to exposure to the mixture. We studied these behaviors by observing fish twice-daily throughout the exposure period at 30-s intervals for 5 min, registering the vertical distribution of fish in the water column and the number of agoniztic acts between all individuals. Fish exposed to the mixture of herbicides were hypoactive and spent more time in the lower parts of the aquaria in comparison to non-exposed controls, reflecting inhibited swimming activity. Average swimming height of exposed fish decreased significantly with the number of agoniztic acts, whilst in control groups there was no significant relationship between the two behaviors. Overall, behavior of fish exposed for a short time to the herbicide mixture was altered in comparison to control-fish behavior. The behavioral endpoints chosen here were easily observed, simple to quantify, and of ecological relevance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study is part of a project aiming to validate the use of the intertidal shanny Lipophorys pholis as a sentinel species in pollution monitoring in NW European marine ecosystems. To this end, a characterisation of acethylcholin (AChE), butyrylcholin (BChE) and propionylcholin (PrChE) esterases in L. pholis muscle was performed and the results indicated that AChE was predominant. Furthermore, the use of eserine sulphate and BW284c51 (0.64-800 microM), and iso-OMPA (0.08-16 mM), confirmed the measurement of true cholinesterases (ChEs) as well as the presence of pseudocholinesterases. The field application of these markers to L. pholis, sampled in seven locations along the Portuguese coast, revealed that fish were likely to be affected by neurotoxic compounds. This was indicated by the significant depletion of AChE (p<0.05) in animals collected at urban and industrialised sites, compared with those from reference locations. The inclusion of a marker of effect, measured as lipid peroxidation levels in muscle tissue, also revealed the existence of site differences. Overall, the study further validates the utility of L. pholis in pollution monitoring studies.

Specimens of Bolinus brandaris (neogastropod) were injected with a single dose of 500 ng/g body weight (b.w.) of tributyltin chloride (TBTCl) or triphenyltin chloride (TPTCl), or a mixture of both compounds (250 ng TBT/g b.w.+250 ng TPT/g b.w.), for a period of up to 31 days. At the end of 4 weeks, significant increases in the female penis size of those gastropods injected with TBT (P<0.05), TPT (P<0.05), or the mixture TBT+TPT (P<0.01) were recorded. In parallel, a group of animals was injected with the neuropeptide APGWamide but this compound failed to promote imposex, suggesting that APGWamide is not involved in imposex promotion in B. brandaris. Acetylcholinesterase activity, a biomarker of neurotoxicity, was determined in the neuroganglia at the end of the experiment, but no significant differences among treatments were found. Overall, these results support the hypothesis that TPT also acts as an endocrine disrupter in this neogastropod species. Our observations also highlight, for the first time, synergistic effects of organotin mixtures having imposex promotion as an endpoint.