Recent development in the field of nanomaterials has given rise into the inquiries regarding the toxicological characteristics of the nanomaterials. While many individual nanomaterials have been screened for their toxicological effects, composites that accompany nanomaterials are not common subjects to such screening through toxicological assessment. One of the widely used composites that accompany nanomaterials is catalyst composite used to reduce air pollution, which was selected as a target composite with nanomaterials for the multifaceted toxicological assessment. As existing studies did not possess any significant data regarding such catalyst composites, this study focuses on investigating toxicological characteristics of catalyst composites from various angles in both in-vitro and in-vivo settings. Initial toxicological assessment on catalyst composites was conducted using HUVECs for cell viability assays, and subsequent in-vivo assay regarding their direct influence on living organisms was done. The zebrafish embryo and its transgenic lines were used in the in-vivo assays to obtain multifaceted analytic results. Data obtained from the in-vivo assays include blood vessel formation, mutated heart morphology, and heart functionality change. Our multifaceted toxicological assessment pointed out that chemical composites augmented with nanomaterials can too have toxicological threat as much as individual nanomaterials do and alarms us with their danger. This manuscript provides a multifaceted assessment for composites augmented with nanomaterials, of which their toxicological threats have been overlooked.

The placenta is essential for sustaining the growth of the fetus. The aim of this study was to investigate the role of the placenta in MCLR-induced significant reduction in fetal weight, especially the changes in placental structure and function. Pregnant mice were intraperitoneally injected with MCLR (5 or 20 μg/kg) from gestational day (GD) 13 to GD17. The results showed MCLR reduced fetal weight and placenta weight. The histological specimens of the placentas were taken for light and electron microscopy studies. The internal space of blood vessels decreased obviously in the placental labyrinth layer of mice treated with MCLR. After the ultrastructural examination, the edema and intracytoplasmic vacuolization, dilation of the endoplasmic reticulum and corrugation of the nucleus were observed. In addition, maternal MCLR exposure caused a reduction of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) expression in placentae, a critical regulator of fetal development. Several genes of placental growth factors, such as Vegfα and Pgf and several genes of nutrient transport pumps, such as Glut1 and Pcft were depressed in placentas of MCLR-treated mice, however nutrient transporters Fatp1 and Snat4 were promoted. Moreover, significant increases in malondialdehyde (MDA) revealed the occurrence of oxidative stress caused by MCLR, which was also verified by remarkable decrease in the glutathione levels, total antioxidant capacity (T-AOC) as well as the activity of antioxidant enzymes. Real-time PCR and western blot analysis revealed that GRP78, CHOP, XBP-1, peIF2α and pIRE1 were remarkable increased in placentas of MCLR-treated mice, indicating that endoplasmic reticulum (ER) stress pathway was activated by MCLR. Furthermore, oxidative stress and ER stress consequently triggered apoptosis which contributed to the impairment of placental development. Collectively, these results suggest maternal MCLR exposure results in reduced fetal body weight, which might be associated with ROS-mediated endoplasmic reticulum stress and impairment in placental structure and function.

Exposure to ZnO-nanoparticles (NPs) in embryonic zebrafish reduces hatching rates which can be mitigated with dissolved organic material (DOM). Although hatching rate can be a reliable indicator of toxicity and DOM mitigation potential, a fish that has been exposed to ZnO-NPs or any other toxicant may also exhibit other abnormal phenotypes not readily detected by the unaided eye. In this study, we moved beyond hatching rate analysis to investigate the consequences of ZnO-NPs exposure on the nervous and vascular systems in developing zebrafish. Zebrafish exposed to ZnO-NPs (1–100 ppm) exhibited an array of cellular phenotypes including: abnormal secondary motoneuron (SMN) axonal projections, abnormal dorsal root ganglion development and abnormal blood vessel development. Dissolved Zn (<10 kDa) exposure also caused abnormal SMN axonal projections, but to a lesser extent than ZnO-NPs. The ZnO-NPs-induced abnormal phenotypes were reversed in embryos concurrently exposed with various types of DOM. In these acute mitigation exposure experiments, humic acid and carbohydrate, along with natural organic matter obtained from the Suwannee River in Georgia and Milwaukee River in Wisconsin, were the best mitigators of ZnO-NPs-induced motoneuron toxicity at 96 h post fertilization. Further experiments were performed to determine if the ZnO-NPs-induced, abnormal axonal phenotypes and the DOM mitigated axonal phenotypes could persist across generations. Abnormal SMN axon phenotypes caused by ZnO-NPs-exposure were detected in F1 and F2 generations. These are fish that have not been directly exposed to ZnO-NPs. Fish mitigated with DOM during the acute exposure (F0 generation) had a reduction in abnormal motoneuron axon errors in larvae of subsequent generations. Therefore, ZnO-NPs exposure results in neurotoxicity in developing zebrafish which can persist from one generation to the next. Mitigation with DOM can reverse the abnormal phenotypes in an acute embryonic exposure context, as well as across generations, resulting in healthy fish.

Forchlorfenuron (CPPU) has been used worldwide, to boost size and improve quality of various agricultural products. CPPU and its metabolites are persistent and have been detected frequently in fruits, water, sediments, and organisms in aquatic systems. Although the public became aware of CPPU through the exploding watermelon scandal of 2011 in Zhenjiang, China, little was known of its potential effects on the environment and wildlife. In this study, adverse effects of CPPU on developmental angiogenesis and vasculature, which is vulnerable to insults of persistent toxicants, were studied in vivo in zebrafish embryos (Danio rerio). Exposure to 10 mg CPPU/L impaired survival and hatching, while development was hindered by exposure to 2.5 mg CPPU/L. Developing vascular structure, including common cardinal veins (CCVs), intersegmental vessels (ISVs) and sub-intestinal vessels (SIVs), were significantly restrained by exposure to CPPU, in a dose-dependent manner. Also, CPPU caused disorganization of the cytoskeleton. In human umbilical vein endothelial cells (HUVECs), CPPU inhibited proliferation, migration and formation of tubular-like structures in vitro. Results of Western blot analyses revealed that exposure to CPPU increased phosphorylation of FLT-1, but inhibited phosphorylation of FAK and its downstream MAPK pathway in HUVECs. In summary, CPPU elicited developmental toxicity to the developing endothelial system of zebrafish and HUVECs. This was do, at least in part due to inhibition of the FAK/MAPK signaling pathway rather than direct interaction with the VEGF receptor (VEGFR).

Microcystin-leucine arginine (MC-LR) has reproductive and developmental toxicities. Previous studies indicated that gestational exposure to MC-LR induced fetal growth restriction in mice. The aim of this study was to further evaluate the effect of paternal MC-LR exposure before mating on fetal development. Male mice were intraperitoneally injected with either normal saline or MC-LR (10 μg/kg) daily for 35 days. Male mouse was then mated with female mice with 1:1 ratio. There was no significant difference on the rates of mating and pregnancy between MC-LR-exposed male mice and controls. Body weight and crown-rump length were reduced in fetuses whose fathers were exposed to MC-LR. Despite no difference on relative thickness of labyrinthine layer, cell proliferation, as measured by Ki67 immunostaining, was reduced in labyrinth layer of MC-LR-exposed mice. Moreover, blood sinusoid area in labyrinth layer was decreased in the fetus whose father was exposed to MC-LR before mating. Correspondingly, cross-sectional area of CD34-positive blood vessel in labyrinth layer was lower in fetuses whose fathers were exposed to MC-LR than in controls. These results provide evidence that paternal MC-LR exposure before mating induces fetal growth restriction partially through inhibiting cell proliferation and vascular development in labyrinth layer.

The aim of this study was to evaluate the acute and chronic effects caused by exposure to the 2,4 dichlorophenoxyacetic acid (2,4-D)-based commercial herbicide Amina Zamba® on Physalaemus albonotatus tadpoles from Gosner stage 25. The lethal concentration (LC50) was determined after exposure to different concentrations of Amina Zamba® (350 to 2400 mg/L) at 96 h. Sublethal effects were evaluated after chronic exposure to four fractions of the LC50₉₆ₕ obtained (12.5, 25, 50, and 75% of LC50₉₆ₕ) and a control. The biological responses analyzed included survival, growth and development, morphological abnormalities, and histological changes in the liver. The LC50 values of Amina Zamba® at 48, 72, and 96 h were 1040.2, 754.2, and 350 mg/L, respectively. The chronic exposure to the herbicide altered the survival of exposed tadpoles and caused several morphological abnormalities and liver histological alterations, mainly at the highest concentrations tested. Oral disc malformations and intestinal abnormalities were the most frequent abnormalities in all treated tadpoles. Histological alterations observed in the liver structure included hepatocyte vacuolization, enlargement of sinusoids, dilation of blood vessels, and a significant increase in the number of melanomacrophages in tadpoles exposed to 25, 50, and 75% LC50₉₆ₕ with respect to control (P < 0.05). Furthermore, the treated tadpoles showed an accelerated development rate, reaching Gosner stages 38 and 42 before controls. These results demonstrate that the chronic exposure to this commercial formulation affects the survival, accelerates metamorphosis, and induces morphological abnormalities and liver damage in P. albonotatus tadpoles.

In this study, the genotoxic and histopathological effects of olive-mill wastewater (OMW) on the tissue cells of Lepomis gibbosus were investigated. The fish were caught from Topçam dam lake (Çine/Aydın) and were exposed to the wastewater in 50-L aquariums which contained 0.5 % OMW for 3–5 and 7 days. In genotoxic investigations, a statistically significant increase was observed in the frequency of micronuclei in the L. gibbosus in experimental groups. As a result of the exposure to OMW, histopathological findings which showed a parallel increase with the amount of exposure in the gill, liver and muscle tissues were determined. In the gills, disruption of lamellae shape, shortening and breakage of primary and secondary lamellae, fusion and branching, separation in the secondary lamellae epithelium, ballooning dilation, hyperplasia in support cells and increase in mucus cells were observed. In the parenchyma of the liver, a difference in local staining, focal necrosis, haemorrhaging in necrotic areas, oedema of blood vessels, expansion in sinusoids, congestion and dilation in portal veins, deterioration of vessel walls, cytoplasmic vacuolization in hepatocytes, pyknotic nuclei, decrease in glycogen storage in hepatocytes near the central vein and aggregates of melanomacrophages were also observed. The necrosis in muscle bundles, widespread oedema between myofibrils, degeneration and separation in some muscle groups, decrease in glycogen content, intramuscular oedema and atrophy in the myofibers were determined in the experimental groups.

The release of pollutants, especially heavy metals, into the aquatic environment is known to have detrimental effects on such an environment and on living organisms including humans when those pollutants are allowed to enter the food chain. The aim of this study is to analyse the damage to Clarias gariepinus’ liver caused by exposure to different concentrations of copper. In the present study, samples of C. gariepinus were exposed to sub-lethal copper sulphate (CuSO₄) concentrations (from 0.2 to 20.0 mg/L) for 96 h. Physiological and behavioural alterations were observed with respect to their swimming pattern, mucus secretion and skin colour. Mortality was also observed at high concentrations of copper. Histopathological alterations of the liver were analysed under light, transmission and scanning electron microscopies. The liver of the untreated group showed normal tissue structures, while histopathological abnormalities were observed in the treated fish under light and electron microscopes with increased copper concentrations. Histopathological abnormalities include necrosis, melanomacrophage, hepatic fibrosis and congested blood vessels. In addition, the enzyme activity of liver cholinesterase (ChE) was also found to be affected by copper sulphate, as 100% of cholinesterase activity was inhibited at 20.0 mg/L. Thus, liver enzyme activity and histopathological changes are proven to be alternative sources for biomarkers of metal toxicity.

Due to the negative effects of pesticide usage in environment, i.e., decline in amphibian populations, this study was designed to examine histopathologic changes following carbaryl exposure for 96 h in the digestive tract of variable toad, Bufotes variabilis. After exposure to single doses of carbaryl (low dose 50 μg/g, medium dose 100 μg/g, and high dose: 200 μg/g), the toads were euthanized and dissected and digestive tract samples were quickly removed. Histopathology revealed esophageal vacuolization and necrosis in esophageal cells. Hemorrhage was also observed in the esophagus. In the stomach, necrosis in the glandular epithelium, congested blood vessels, edema among gastric glands, dilated tips of the mucosal epithelial layer, gastric glandular atrophy, and hemorrhage were found. In the intestine, edema within villi, hemorrhage, inflammation, vacuolization, and necrosis in epithelial cells of villi were detected. This study clearly showed that carbaryl caused adverse effects on the digestive tract of B. variabilis in all dose groups.

Nanotechnology is a novel arena with promising applications in the field of medicine, industry, and agriculture including fisheries. Cross-disciplinary interactions and the application of this technology in biological systems have led to the innovation of novel nanoparticle antioxidants, which are the subject of our study. In context with above background, we designed an experiment on nano-silver to elucidate its role for mitigation of abiotic and biotic stress. Three diets were formulated viz. silver nanoparticles (Ag-NPs) incorporated at 0.5 and 1 mg/kg diet and control diet (Ag-NPs at 0 mg/kg). Fish were exposed to sublethal level of 1/25th of LC₅₀ (4 ppm) of lead (Pb) and temperature at 34 °C. The effect of Ag-NPs on productive performance (weight gain %, feed conversion ratio, protein efficiency ratio, and specific growth rate), stress biomarkers (catalase, super oxide dismutase, glutathione-s-transferase, acetylcholine esterase, cortisol, heat shock protein), biochemical and immunological response (protein and carbohydrate metabolic enzymes, phagocytic activity, serum total protein and albumin: globulin ratio), histopathology alterations in the liver and gill as well as survival of Channa striatus, following challenge with pathogenic bacteria were evaluated. Dietary Ag-NPs at 0.5-mg supplementation improved growth performance, immunity, survival, and reduced stress biomarker such as HSP 70, cortisol, and blood glucose in various fish tissues. Exposure to Pb and high temperature and group fed with Ag-NPs (1 mg/kg diet) demonstrated remarkable changes in the histo-architect of liver such as pyknotic nuclei, pyknosis, leucocyte infiltration, hemorrhage and karyokinesis, blood vessels with nucleated, lipid vacuoles in the liver tissue. Histology of gill displayed hyperplasia, aneurism, blood congestion, severe telengiectiasis, epithelial lifting, curling of secondary lamella, hyperplasia of epithelial cell of secondary lamella in the group exposed to lead and high temperature and supplemented with Ag-NPs at 1 mg/kg diet. In addition to histopathology, feeding with Ag-NPs at 1 mg/kg diet deteriorated and altered all studied parameters including reduced growth performance. Results obtained in the present study suggest that supplementation of Ag-NPs at 0.5 mg/kg diet has a definitive role to play in the mitigation of abiotic and biotic stress in C. striatus.