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Multicollinearity, characterized by significant co-expression patterns among genes, often occurs in high-throughput expression data, potentially impacting the predictive model's reliability. This study examined multicollinearity among closely related genes, particularly in RNA-Seq data obtained from embryoid bodies (EB) exposed to 5-fluorouracil perturbation to identify genes associated with embryotoxicity. Six genes-Dppa5a, Gdf3, Zfp42, Meis1, Hoxa2, and Hoxb1-emerged as candidates based on domain knowledge and were validated using qPCR in EBs perturbed by 39 test substances. We conducted correlation studies and utilized the variance inflation factor (VIF) to examine the existence of multicollinearity among the genes. Recursive feature elimination with cross-validation (RFECV) ranked Zfp42 and Hoxb1 as the top two among the seven features considered, identifying them as potential early embryotoxicity assessment biomarkers. As a result, a t test assessing the statistical significance of this two-feature prediction model yielded a p value of 0.0044, confirming the successful reduction of redundancies and multicollinearity through RFECV. Our study presents a systematic methodology for using machine learning techniques in transcriptomics data analysis, enhancing the discovery of potential reporter gene candidates for embryotoxicity screening research, and improving the predictive model's predictive accuracy and feasibility while reducing financial and time constraints.

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Individuals are exposed to a wide arrays of hazardous chemicals on a daily basis through various routes, many of which have not undergone comprehensive toxicity assessments. While traditional developmental toxicity tests involving pregnant animals are known for their reliability, they are also associated with high costs and time requirements. Consequently, there is an urgent demand for alternative, cost-efficient, and rapid in vitro testing methods. This study aims to address the challenges related to automating and streamlining the screening of early developmental toxicity of chemicals by introducing a mouse embryoid body test (EBT) model in a 384-ultra low attachment well format. Embryoid bodies (EBs) generated in this format were characterized by a spontaneous differentiation trajectory into cardiac mesoderm by as analyzed by RNA-seq. Assessing prediction accuracy using reference compounds suggested in the ICH S5(R3) guideline and prior studies resulted in the establishment of the acceptance criteria and applicability domain of the EBT model. The results indicated an 84.38% accuracy in predicting the developmental toxicity of 23 positive and 9 negative reference compounds, with an optimized cutoff threshold of 750 µM. Overall, the developed EBT model presents a promising approach for more rapid, high-throughput chemical screening, thereby facilitating well-informed decision-making in environmental management and safety assessments.

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Background Herbal medicine has gathered increasing attention in contemporary healthcare practices, offering natural remedies for a wide range of ailments such as skin diseases, liver disorders, bronchitis, and asthma. Among the plethora of medicinal plants, Croton bonplandianum, commonly known as "Ban Tulsi," holds significant medicinal value owing to its diverse pharmacological properties. This study investigated the cytotoxicity, embryotoxicity, antidiabetic, and anticariogenic effects of an ethanolic extract derived from C. bonplandianum. The research objectives were to explore the preparation of an ethanolic extract of C. bonplandianum and employ a multifaceted approach by evaluating its cytotoxicity, embryotoxicity, anticariogenic, and antidiabetic potentials. Materials and methods In this study, the ß-glucosidase inhibitory and the α-amylase inhibitory assays were utilized to evaluate the antidiabetic activity of the C. bonplandianum ethanolic extract. The in vitro cytotoxicity activity was assessed by using the brine shrimp lethality assay (BSLA), and embryotoxicity was evaluated using zebrafish embryos and larvae. Through the agar well diffusion method and the time-kill curve analysis, the anticariogenic activity was evaluated. Results In α-amylase and ß-glucosidase inhibitory assays, the ethanolic extract of C. bonplandianum showed potent antidiabetic properties, near those of standard acarbose. The cytotoxicity evaluation using the BSLA showed less toxicity. The anticariogenic activity of the ethanolic extract of C. bonplandianum was assessed by comparing the standard (Amoxyrite) in terms of its zone of inhibition against oral pathogens such as Streptococcus mutans and Lactobacillus species (spp.). The antibacterial efficiency was validated using a time-kill curve assay in which the study depends on the concentration of the bacterial pathogenic organisms, namely, Lactobacillus spp. and S. mutans. In embryotoxicity evaluation, there were no morphological malformations in zebrafish larvae or embryos when exposed to high concentrations of C. bonplandianum ethanolic extract. Conclusion The ethanolic extract of C. bonplandianum exhibited promising antidiabetic and anticariogenic effects, supporting its conventional usage in alternative medicine. The outcomes of these research analyses suggest the plant potential as a natural source of compounds with bioactive qualities and can be utilized in the healthcare and pharmaceutical industries.

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Increasing global demands for oils are fueling the production of diluted bitumen (DB) from Canada's oil sands region. More weathered than conventional crude (CC) oils, Alberta bitumen is often diluted with lighter petroleum oils to reduce density and viscosity to meet pipeline specifications for transportation. Being a heavy oil product that is transported in large volumes across Canada and the USA, there has been interest to compare its behavior and toxicity characteristics when spilled to those of CC. To determine the influence of environmental weathering upon DB following a freshwater spill, we conducted separate controlled spills of Cold Lake Blend DB and Mixed Sweet Blend light CC oil in a mesocosm spill-tank system at 24 °C with wave-action for 56 days. DB-contaminated waters remained acutely lethal for a period of 14 days to early life stage fathead minnows (Pimephales promelas) exposed during embryologic development, while CC was lethal for 1 day. However, concentrations of mono- and polycyclic aromatic compounds, often claimed to be principally responsible for the acute and chronic toxicity of crude oils, were consistently higher in CC water compared to DB. Elevated aromatic concentrations in CC water correlated with higher prevalences of developmental malformations, reduced heart and growth rates, and impacts on the aryl hydrocarbon receptor pathway. Organic acids were measured over the course of the studies and O2 containing naphthenic acids were present at greater relative abundances in DB- compared to CC-contaminated water, with their attenuation correlating with reduced acute and sublethal toxicity. Furthermore, organic acid degradation products accumulated with time and likely contributed to the consistently sublethal toxicity of the weathered oils throughout the experiment. Improved characterization of the fractions including organic acids and those organic compounds found within the unresolved complex mixture of fresh and weathered crude oils is necessary to adequately understand and prepare for the risks that accidental petroleum spills pose to aquatic resources.

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Drug use during pregnancy is an important issue that must be investigated due to its adverse effects on maternal and foetal health. This study aimed to determine the embryotoxic and teratogenic effects of in-ovo administered metamizole (dipyrone), which can be used when needed during pregnancy and has potent analgesic, antipyretic, anti-inflammatory, and long bone (tibia and femur) effects. This study used 240 fertile eggs from Atak S breed chickens, divided into eight equal groups: control, vehicle control, and 15.62, 31.25, 62.5, 125, 250 and 500 mg/kg metamizole. The eggs were hatched on the 21st day of incubation, and the chicks' body weights and mortality rates were determined. The right and left femur and tibia bones were resected from the chicks. Anatomical reference points were determined after removing the soft tissues of the bones, and necessary morphometric measures were taken from these points with a 0.01 mm precision using digital callipers. The 100% lethal dose (LD100) was identified in the highest examined dose (500 mg/kg) in the Chicken Embryotoxicity Screening Test (CHEST)-I stage. The CHEST-II stage determined the 50% lethal dose (LD50). High-dose metamizole affected skeletal development, significantly decreasing tibia and femur lengths and corpus thicknesses and increasing mortality.

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Benzophenones (BPs) are widely used as photoinitiators (PIs) or printing inks in food packaging, which may migrate into foods. However, the toxicity information of some BP analogues, such as 4,4'-bis(diethylamino)-benzophenone (DEAB), 4-phenylbenzophenone (4-PBP), 4 (hydroxymethyl)benzophenone (4-HMBP), those are used as PIs is lacking. Developmental toxicity is a health concern associated with PIs exposure. Recently, alternative non-in vivo methods have been proposed to evaluate the concerned chemicals or better understand the modes of action of certain toxicological endpoints. In this study, using in silico methods, we predicted that BP, DEAB, 4-PBP and 4-HMBP might exhibit developmental toxicity. However, we found that only DEAB is strong embryotoxic and disturbs the early differentiation of mouse embryonic stem cells into three germ layers and cardiomyocytes. DEAB treatment also prevented cardiomyocyte differentiation in human induced pluripotent stem cells (hiPSCs) on day 10. However, BP, 4-PBP and 4-HMBP had no similar effects on cardiomyocyte differentiation on day 10. Transcriptomic analysis revealed that treatment with DEAB significantly decreased the mRNA levels of differentiation-related transcription factors SOX17 and FOXA1, in hiPSCs on day 4. Furthermore, DEAB treatment caused tail malformations and yolk sac edema in zebrafish embryos. To conclude, DEAB may be embryotoxic because it disturbs the early differentiation of stem cells. Further studies are warranted to better understand the health effects of DEAB exposure.

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The frequent occurrence of antibiotics in the aquatic environment has engendered negative impacts on non-target organisms. The effects of the veterinary antibiotic florfenicol (FLO) during the embryo-larval development of the sea urchin, Paracentrotus lividus was assessed using four increasing concentrations (1, 2, 5 and 10 mg/L). Furthermore, FLO toxicity to adults was investigated through the analysis of oxidative damage, histopathological alterations, lipid metabolism and acetylcholinesterase activity following an exposure period of 96 h. FLO induced embryotoxicity with estimated EC50 values of 5.75, 7.56 and 3.29 mg/L after 12 h, 24 h and 48 h, respectively. It generated oxidative stress assessed as lipid peroxidation in gonads despite the increased antioxidant activity of catalase (CAT). Neurotoxicity was also evident since the AChE activity significantly decreased. Moreover, FLO affected the lipid metabolism by increasing saturated fatty acid (SFA) and monounsaturated fatty acid proportions (MUFA), except in the group exposed to 5 mg/L. The increase in polyunsaturated fatty acid (PUFA) levels and docosahexaenoic acid (DHA, C22:6n-3) proportions were noted with all FLO concentrations. Eicosapentaenoic acid (EPA, C20:5n-3) decreased, while arachidonic acid (ARA, C20:4n-6) increased in sea urchins exposed to 5 and 10 mg/L FLO. Histopathological alterations of gonadal tissues represent an additional confirmation about the toxicity of this antibiotic that might decrease the reproductive performance of this species. Nevertheless, even if reproduction of sea urchins would be partially successful, the embryotoxicity would compromise the normal development of the embryos with consequences on the population.

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Thiacloprid, a hazardous neonicotinoid insecticide, prevalent in daily agricultural practices, raises concerns due to the harmful effects of its residues on food items, and on unintended organisms poses a significant threat to human health. Introduced in 1990, Thiacloprid have gained popularity for its perceived effectiveness and reduced risks to non-target animals. However, emerging research in recent years reports significant toxic effects of Thiacloprid on non-target species, spanning neurotoxicity, immunotoxicity, hepatotoxicity, nephrotoxicity, and reproductive issues. Mammalian studies, particularly involving rodents, reveal cognitive impairment, hippocampal damage, and hepatic abnormalities upon Thiacloprid exposure. Reproductive toxicity and DNA damage are imminent concerns, disrupting gestational epigenetic reprogramming and suggesting persistent effects on future generations. Genotoxic effects, Embryotoxic, and observed reproductive toxicity accentuate the need for caution in the utilization of Thiacloprid. This review highlights reported toxic effects produced by Thiacloprid in recent years, challenging the initial belief in its lower toxicity for vertebrates.

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Organophosphorus flame retardants (OPFRs) such as triphenyl phosphate (TPHP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) were reported to impair cardiac function in fish. However, limited information is available regarding their cardiotoxic mechanisms. Using rare minnow (Gobiocypris rarus) as a model, we found that both TPHP and TDCIPP exposures decreased heart rate at 96 h postfertilization (hpf) in embryos. Atropine (an mAChR antagonist) can significantly attenuate the bradycardia caused by TPHP, but only marginally attenuated in TDCIPP treatment, suggesting that TDCIPP-induced bradycardia is independent of mAChR. Unlike TDCIPP, although TPHP-induced bradycardia could be reversed by transferring larvae to a clean medium, the inhibitory effect of AChE activity persisted compared to 96 hpf, indicating the existence of other bradycardia regulatory mechanisms. Transcriptome profiling revealed cardiotoxicity-related pathways in treatments at 24 and 72 hpf in embryos/larvae. Similar transcriptional alterations were also confirmed in the hearts of adult fish. Further studies verified that TPHP and TDCIPP can interfere with Na+/Ca2+ transport and lead to disorders of cardiac excitation-contraction coupling in larvae. Our findings provide useful clues for unveiling the differential cardiotoxic mechanisms of OPFRs and identifying abnormal Na+/Ca2+ transport as one of a select few known factors sufficient to impair fish cardiac function.

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The aim of the present work is to propose a new quantitative assessment method (FETAX-score) for determining the degree of Xenopus laevis embryo development intended for use in embryotoxicity studies. Inspired by a similar scoring system used to evaluate developmental delays (young-for-age phenotypes) in rat embryos cultured in vitro, the FETAX-score was established by considering seven morphological features (head, naris, mouth, lower jaw, tentacles, intestine, anus) that are easily evaluable in tadpoles during the late stages of development at the conclusion of the test. Given that X. laevis development is temperature-dependent and that temperatures below 14°C and above 26°C are teratogenic, the FETAX-score was tested in embryos maintained at 17, 20, 23 and 26°C. No abnormalities were observed in any group, while the total score was temperature-related, suggesting that the FETAX-score is sensitive to moderate distress that does not influence general morphology. Intestine and anus were the least sensitive structures to temperature variations. To assess the applicability of the FETAX-score in developmental toxicological studies, we evaluated FETAX-score in tadpoles exposed during the morphogenetic period to Ethanol (Eth) at concentrations of 0, 0.25, 0.5, 1, 1.5, and 2 % v/v. Gross malformations were observed only in tadpoles from the Eth 2 % group. By contrast, data analysis of the other Eth groups showed dose-related reductions in the FETAX-score. Tentacles were the most sensitive structures to Eth-related delays. These results support the use of the FETAX-score to quantitatively assess developmental deviations in FETAX embryotoxicity studies.

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This study aimed to evaluate the environmental impact of dental materials: commercial composite Tetric EvoCeram®, glass ionomer Equia Forte® HT Fil, laboratory-prepared composite, alkasite Cention® Forte, amalgam Amalcap® Plus, and samples from dental chair drainage systems (DCDS). Methacrylate monomers were detected in the eluates of experimental and commercials composites, and alkasite. In DCDS samples solely mercury was found at concentrations of 0.08-1.86 µg/L. The experimental composite (48 h incubation) exhibited the highest toxicity on zebrafish Danio rerio (LC50=0.70 g/L), followed by amalgam (LC50=8.27 g/L) < Tetric EvoCeram® (LC50=10.94 g/L) < Equia Forte® HT Fil (LC50=24.84 g/L) < Cention® Forte (LC50=32.22 g/L). Exposure of zebrafish to DCDS samples resulted in decreased larval body length and increased occurrences of edema and blood accumulation. The results obtained highlight the need for additional monitoring and further research on the release of unreacted monomers and mercury from dental materials and their environmental impact.

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The Villa Victoria dam is one of the most important storage reservoirs in Mexico since it distributes water to more than 20 million inhabitants in the Metropolitan Zone of Mexico City. In this dam, the common carp (Cyprinus carpio) is an important food resource for the inhabitants, so the aim of this work was to evaluate the oxidative damage (lipoperoxidation, oxidized proteins, antioxidant enzymes activity and gene expression), AChE, embryotoxicity and behavioral changes in C. carpio embryos and larvae exposed to water from Villa Victoria dam for 24, 48, 72 and 96 h. The embryotoxicity was evaluated trough the General Morphology Score (GMS) and the teratogenic index. Behavioral changes in basal locomotor activity and thigmotaxis were evaluated in a DanioVision, Noldus ™. An increase in lipid and protein oxidation as well as modification of CAT, SOD and GPx enzymatic activity was observed during the exposure times. The GMS indicated a low development in the embryos, the teratogenic index was less than 1, however teratogenic effects as yolk edema, fin malformation, head malformation and scoliosis were observed. In parallel, an increase in AChE activity and gene expression was observed reflecting changes in distance traveled of the basal locomotor activity and thigmotaxis at the sampling points. In conclusion, pollutants in water from Villa Victoria dam caused oxidative damage, changes in SOD, CAT, GPx and AChE activity as well as embryotoxicity and modifications in the behavior of C. carpio larvae. This study demonstrates the need to implement restoration programs for this reservoir since, contamination in the Villa Victoria dam could eventually endanger aquatic life and human health.

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Due to its endocrine disruptive activity, the plastic additive Bisphenol A (BPA) is classified as substance of very high concern (EU ECHA 2017). A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species including the amphibian Xenopus laevis, where severe branchial defects were associated to lethality. The exposure of X. laevis embryos to the BPA analogue bisphenol B (BPB) was recently linked to similar teratogenic effects, with BPB having relative potency about 3 times higher than BPA. The combined BPA-BPB exposure is realistic as both BPA and BPB are detected in human samples and environment. Limited experimental data are available on the combined developmental toxicity of BPA and BPB. The aim of the present work is to evaluate the effects of BPA and BPB mixture in the X. laevis development model, using R-FETAX procedure. The exposure was limited to the first day of development (corresponding to the phylotypic developmental period, common to all vertebrates). Samples were monitored for lethal effects during the full six-day test period and the external morphology was evaluated at the end of the test. Mixture effects were described by modelling, using the PROAST software package. Overall data modelling showed that dose-addiction could not be rejected, suggesting a health concern for co-exposure.

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Gold complexes can be a useful system in the fight against cancer. Although many studies have been carried out on in vitro 2D cell culture models embryotoxic assays are particularly lacking. Embryotoxicity and DNA damage are critical concerns in drug development. In this study, the effects of a new N-Heterocyclic carbene (NHC)-Au compound (Bromo[1,3-di-4-methoxybenzyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I)) at different concentrations were explored using multifaceted approach, encompassing 2D cancer cell cultures, in vivo zebrafish and in vitro bovine models, and compared with a consolidated similar complex (Bromo[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I)). The results obtained from 2D cancer cell cultures revealed concentration-dependent effects of the gold compounds by estimating the cytotoxicity with MTT assay and cellular damage as indicated by LDH release. Selected concentrations of gold complexes demonstrated no adverse effects on zebrafish embryo development. However, in bovine embryos, these same concentrations led to significant impairments in the early developmental stages, triggering cell apoptosis and reducing blastocyst competence. These findings underscore the importance of evaluating drug effects across different model systems to comprehensively assess their safety and potential impact on embryonic development.

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OBJECTIVES: Composites with copper-doped mesoporous bioactive nanospheres (Cu-MBGN) were developed to prevent secondary caries by imparting antimicrobial and ion-releasing/remineralizing properties. METHODS: Seven experimental composites containing 1, 5 or 10 wt% Cu-MBGN, the corresponding inert controls (silica) and bioactive controls (bioactive glass 45S5) were prepared. The temperature rise during light curing, cross-linking density by ethanol softening test, monomer elution and their potential adverse effects on the early development of zebrafish Danio rerio was investigated. RESULTS: Materials combining Cu-MBGN and silica showed the highest resistance to ethanol softening, as did the bioactive controls. Cu-MBGN composites showed significant temperature rise and reached maximum temperature in the shortest time. Bisphenol A was not detected, while bis-GMA was found only in the control materials and TEGDMA in the eluates of all materials. There was no increase in zebrafish mortality and abnormality rates during exposure to the eluates of any of the materials. CONCLUSIONS: The composite with 5 wt% Cu-MBGN combined with nanosilica fillers showed the lowest ethanol softening, indicating the polymer's highest durability and cross-linking density. Despite the TEGDMA released from all tested materials, no embryotoxic effect was observed.

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Background/aim: This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. Materials and methods: A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. Results: No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). Conclusion: It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

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Many marine organisms possess an essential capacity to produce secondary metabolites that exhibit toxic characteristics. A new polyhydroxy steroid, 24-methyl-5α-cholestane-24(28)-ene-3ß, 4ß, 6α, 7α, 8, 15ß, 16ß, 26-octol-6-O-sodium sulphate (1) was isolated from starfish (Asterina pectinifera), along with five polar steroid compounds (2-6) that were previously identified. NMR (1H and 13C NMR, 1H-1H COSY, HSQC, HMBC, and NOESY) and HR-ESI-MS were employed for structure elucidations. The embryotoxicity and teratogenicity of the isolated compounds were assessed using embryos of marine medaka (Oryzias melastigma). Compound 5 exhibited moderate embryotoxicity (96h-LC50: 65 µM).

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In the framework of a safe-by-design approach, we previously assessed the eco-safety of nanostructured cellulose sponge (CNS) leachate on sea urchin reproduction. It impaired gamete quality, gamete fertilization competence, and embryo development possibly due to the leaching of chemical additives used during the CNS synthesis process. To extend this observation and identify the component(s) that contribute to CNS ecotoxicity, in the present study, we individually screened the cytotoxic effects on sea urchin Arbacia lixula and Paracentrotus lividus gametes and embryos of the three main constituents of CNS, namely cellulose nanofibers, citric acid, and branched polyethylenimine. The study aimed to minimize any potential safety risk of these components and to obtain an eco-safe CNS. Among the three CNS constituents, branched polyethylenimine resulted in the most toxic agent. Indeed, it affected the physiology and fertilization competence of male and female gametes as well as embryo development in both sea urchin species. These results are consistent with those previously reported for CNS leachate. Moreover, the characterisation of CNS leachate confirmed the presence of detectable branched polyethylenimine in the conditioned seawater even though in a very limited amount. Altogether, these data indicate that the presence of branched polyethylenimine is a cause-effect associated with a significant risk in CNS formulations due to its leaching upon contact with seawater. Nevertheless, the suggested safety protocol consisting of consecutive leaching treatments and conditioning of CNS in seawater can successfully ameliorate the CNS ecotoxicity while maintaining the efficacy of its sorbent properties supporting potential environmental applications.

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Green silver nanoparticles (G-Ag NPs) have contributed to the development of ecological technologies with low environmental impact and safer for human health, as well as demonstrating potential for the control of vectors and intermediate hosts. However, knowledge about its toxicity in the early stages of gastropod development remains scarce. Therefore, the current study aimed to investigate the toxicity of G-Ag NPs synthesized from Croton urucurana leaf extracts in snail species Biomphalaria glabrata, which is an intermediate host for Schistosoma mansoni parasite. G-Ag NPs were synthesized using two types of plant extracts (aqueous and hydroethanolic) and characterized using multiple techniques. Bioassays focused on investigating G-Ag NPs and plant extracts were carried out with embryos and newly hatched snails, for 144 h and 96 h, respectively; toxicity was analyzed based on mortality, hatching, development inhibition, and morphological changes. Results have shown that both G-Ag NPs were more toxic to embryos and newly hatched snails than the investigated plant extracts. G-Ag NPs deriving from aqueous extract have higher molluscicidal activity than those deriving from hydroethanolic extract. Both G-Ag NPs induced mortality, hatching delay, development inhibition, and morphological changes (i.e., hydropic embryos), indicating their molluscicidal activities. Moreover, embryos were more sensitive to G-Ag NPs than newly hatched snails. Thus, the toxicity of G-Ag NPs to freshwater snails depends on the type of extracts and the snail's developmental stages. These findings can contribute to the development of green nanobiotechnologies applicable to control snails of medical importance.

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Triclopyr, an auxin-like herbicide that is widely employed for managing weeds in food crops and pastures, has been identified in various environmental settings, particularly aquatic ecosystems. Limited understanding of the environmental fate of this herbicide, its potential repercussions for both the environment and human health, and its insufficient monitoring in diverse environmental compartments has caused it to be recognized as an emerging contaminant of concern. In this study, we have investigated how triclopyr affects zebrafish, considering a new alternative methodology. We focused on the endpoints of developmental toxicity, neurotoxicity, and behavior of zebrafish embryos and larvae. We determined that triclopyr has a 96 h median lethal concentration of 87.46 mg/L (341.01 µM). When we exposed zebrafish embryos to sublethal triclopyr concentrations (0.5, 1, 5, 10, and 50 µM) for up to 144 h, we found that 50 µM triclopyr delayed zebrafish egg hatchability. Yolk sac malabsorption was significant at 0.5, 1, 5, and 10 µM triclopyr. In zebrafish larvae, uninflated swim bladder was significant only at 50 µM triclopyr. Furthermore, zebrafish larvae had altered swimming activity after exposure to 10 µM triclopyr for 144 h. In summary, these comprehensive results indicate that even low triclopyr concentrations can elicit adverse effects during early zebrafish development.