Toxicity of Titanium Dioxide-Cerium Oxide Nanocomposites to Zebrafish Embryos: A Preliminary Evaluation.

The widespread use of metal nanoparticles in different fields has raised many doubts regarding their possible toxicity to living organisms and the accumulation and discharge of metals in fish species. Among these nanoparticles, titanium dioxide (TiO2) and cerium oxide (CeO2) nanoparticles have mainly been employed in photocatalysis and water depuration. The aim of this research was to evaluate the potential toxic effects, after a co-exposure of TiO2-3%CeO2 nanoparticles, on zebrafish development, using an acute toxicity test. Increasing concentrations of TiO2-3%CeO2 nanoparticles were used (0.1-1-10-20 mg/L). The heartbeat rate was assessed using DanioscopeTM software (version 1.2) (Noldus, Leesburg, VA, USA), and the responses to two biomarkers of exposure (Heat shock proteins-70 and Metallothioneins) were evaluated through immunofluorescence. Our results showed that the co-exposure to TiO2-3%CeO2 nanoparticles did not affect the embryos' development compared to the control group; a significant difference (p < 0.05) at 48 hpf heartbeat for the 1, 10, and 20 mg/L groups was found compared to the unexposed group. A statistically significant response (p < 0.05) to Heat shock proteins-70 (Hsp70) was shown for the 0.1 and 1 mg/L groups, while no positivity was observed in all the exposed groups for Metallothioneins (MTs). These results suggest that TiO2-3%CeO2 nanocomposites do not induce developmental toxicity; instead, when considered separately, TiO2 and CeO2 NPs are harmful to zebrafish embryos, as previously shown.

Polystyrene Nanoplastics in Aquatic Microenvironments Affect Sperm Metabolism and Fertilization of Mytilus galloprovincialis (Lamark, 1819).

The continuous and unregulated discharge of wastes and pollutants into the aquatic environment has required constant monitoring of the risks incurred by aquatic ecosystems. Alarmism arises from plastic pollution as larger artifacts release nanoscale fragments that can contact free-living stages such as gametes, embryos, and larvae. Specifically, the interaction between spermatozoa, released in water in externally fertilizing species, and the surrounding microenvironment is essential for successful fertilization. Activation and kinematics of movement, proper maintenance of ionic balance, and chemotactism are processes highly sensitive to even minimal perturbations caused by pollutants such as polystyrene nanoplastics. Spermatozoa of Mytilus galloprovincialis (M. galloprovincialis), an excellent ecotoxicological model, undergo structural (plasma membrane ruptures, DNA damage) and metabolic (reduced motility, fertilizing capacity) damage upon exposure to 50 nm amino-modified polystyrene nanoplastics (nPS-NH2). Nanoplastics of larger diameter (100 nm) did not affect sperm parameters. The findings highlighted the negative impact that plastic pollution, related to nanoparticle diameter and concentration, could have on sperm quality and reproductive potential of organisms, altering the equilibrium of aquatic ecosystems.

Sublethal Effects of Polystyrene Nanoplastics on the Embryonic Development of Artemia salina (Linnaeus, 1758).

Currents, wave motion, solar radiation, and abrasion are mechanisms responsible for the degradation of large plastic artifacts and contribute to the dispersion of micro and nanoplastics into aquatic ecosystems, which are, currently, the most dangerous threats due to their invisibility and persistence. The present work evaluated the possible lethal and sublethal effects of amino-modified polystyrene nanoplastics (nPS-NH2) with diameters of 50 nm and 100 nm on Artemia salina (A. salina), an organism at the base of the trophic chain of the aquatic system, using a widely used model for the analysis of embryotoxicity from environmental pollutants. For this purpose, after evaluating the biodistribution of nanoplastics in the body of the tested animals, several endpoints such as anomalies, apoptosis, and ROS production were assessed. In addition, particular attention was dedicated to evaluating the correlation between toxicity and the particle size tested. The results reported that, despite the absence of a lethal impact, several sublethal effects involving gut and body size malformations, as well as the enhancement of apoptosis and oxidative stress in relation to an increase in tested concentration and a decrease in nanoparticle size.

Titanium Dioxide Nanoparticles: Effects on Development and Male Reproductive System.

Titanium dioxide nanoparticles (TiO2-NPs) are used intensively. Thanks to their extremely small size (1-100 nm), TiO2-NPs are more absorbable by living organisms; consequently, they can cross the circulatory system and then be distributed in various organs including the reproductive organs. We have evaluated the possible toxic effect of TiO2-NPs on embryonic development and the male reproductive system using Danio rerio as an organism model. TiO2-NPs (P25, Degussa) were tested at concentrations of 1 mg/L, 2 mg/L, and 4 mg/L. TiO2-NPs did not interfere with the embryonic development of Danio rerio, however, in the male gonads the TiO2-NPs caused an alteration of the morphological/structural organization. The immunofluorescence investigation showed positivity for biomarkers of oxidative stress and sex hormone binding globulin (SHBG), both confirmed by the results of qRT-PCR. In addition, an increased expression of the gene responsible for the conversion of testosterone to dihydrotestosterone was found. Since Leydig cells are mainly involved in this activity, an increase in gene activity can be explained by the ability of TiO2-NPs to act as endocrine disruptors, and, therefore, with androgenic activity.

Effects of carnosine on the embryonic development and TiO2 nanoparticles-induced oxidative stress on Zebrafish.

Oxidative stress is due to an unbalance between pro-oxidants, such as reactive oxygen (ROS) and nitrogen (RNS) species, and antioxidants/antioxidant system. Under physiological conditions these species are involved in different cellular processes such as cellular homeostasis and immune response, while an excessive production of ROS/RNS has been linked to the development of various diseases such as cancer, diabetes, and Alzheimer's disease. In this context, the naturally occurring dipeptide carnosine has shown the ability to scavenge ROS, counteract lipid peroxidation, and inhibit proteins oxidation. Titanium dioxide nanoparticles (TiO2-NPs) have been widely used to produce cosmetics, in wastewater treatment, in food industry, and in healthcare product. As consequence, these NPs are often released into aquatic environments. The Danio rerio (commonly called zebrafish) embryos exposure to TiO2-NPs did not affect the hatching rate, but induced oxidative stress. According to this scenario, in the present study, we first investigated the effects of carnosine exposure and of a sub-toxic administration of TiO2-NPs on the development and survival of zebrafish embryos/larvae measured through the acute embryo toxicity test (FET-Test). Zebrafish larvae represent a useful model to study oxidative stress-linked disorders and to test antioxidant molecules, while carnosine was selected based on its well-known multimodal mechanism of action that includes a strong antioxidant activity. Once the basal effects of carnosine were assessed, we then evaluated its effects on TiO2-NPs-induced oxidative stress in zebrafish larvae, measured in terms of total ROS production (measured with 2,7-dichlorodihydrofluorescein diacetate probe) and protein expression by immunohistochemistry of two cellular stress markers, 70 kDa-heat shock protein (Hsp70) and metallothioneins (MTs). We demonstrated that carnosine did not alter the phenotypes of both embryos and larvae of zebrafish at different hours post fertilization. Carnosine was instead able to significantly decrease the enhancement of ROS levels in zebrafish larvae exposed to TiO2-NPs and its antioxidant effect was paralleled by the rescue of the protein expression levels of Hsp70 and MTs. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of pathologies characterized by oxidative stress such as neurodegenerative disorders.

In Vitro Nano-Polystyrene Toxicity: Metabolic Dysfunctions and Cytoprotective Responses of Human Spermatozoa.

The ubiquitous spread of Polystyrene nanoplastics (PS-NPs) has rendered chronic human exposure an unavoidable phenomenon. The biodistribution of such particles leads to bioaccumulation in target organs including the testis, the site of sperm maturation. The purpose of this research has been to estimate the impact of PS-NPs (50 and 100 nm) on the metabolism of mature spermatozoa. The analysis of the semen parameters has revealed a higher toxicity of the smaller sized PS-NPs, which have negatively affected major organelles, leading to increased acrosomal damage, oxidative stress with the production of ROS, DNA fragmentation, and decreased mitochondrial activity. PS-NPs of 100 nm, on the other hand, have mainly affected the acrosome and induced a general state of stress. An attempt has also been made to highlight possible protective mechanisms such as the expression of HSP70s and their correlation among various parameters. The results have evinced a marked production of HSP70s in the samples exposed to the smaller PS-NPs, negatively correlated with the worsening in oxidative stress, DNA fragmentation, and mitochondrial anomalies. In conclusion, our results have confirmed the toxicity of PS-NPs on human spermatozoa but have also demonstrated the presence of mechanisms capable of counteracting at least in part these injuries.

Antioxidant activity of two Opuntia Mill. species fruit extracts on human sperm quality after a freeze-thaw cycle.

This study investigated the phenolic compounds and antioxidant capacity of fruit extracts of Opuntia dillenii (Ker Gawl.) Haw.(OD) and Opuntia ficus-indica (L.) Mill.(OFI), yellow (F1) and red (F2) varieties. In order to evaluate the antioxidant activity of these extracts on human sperm quality after thawing, the semen parameters (vitality, motility, acrosome reaction, oxidative stress, and DNA fragmentation) were analysed after 1 h of exposure. The results showed that OD has higher phenolic content and antioxidant power than OFI, and that they are higher in F2 than F1. Furthermore, regarding the activity of extracts on thawed sperm, the results showed a significant increase in motility in samples treated with OFI F1 and OD extracts, while an improvement in vitality and acrosome reaction and a reduction of DNA fragmentation were observed in all exposed samples compared to the control. Finally, a reduction of oxidative stress was observed in samples exposed to OFI F2 and OD than control.

Acute Toxicity of a Marine Emerging Pollutant (Promethazine Hydrochloride) on Artemia sp.

Recently, there has been a worrying increase in the pollution of the aquatic ecosystem caused by emerging contaminants (ECs) detected in wastewater effluent discharges. Although traces of ECs in waters have been found in low concentrations, it leads to negative effects for nontarget organisms. Antihistamines are a class of drugs largely used, whose metabolites are widespread in the aquatic ecosystem. The aim of the study was to evaluate the short-term effects of promethazine hydrochloride on nauplii of Artemia sp. A high percentage of mortality and morphological alterations were found. The results suggest a possible correlation between exposure to antihistamine and an acceleration of larval development.

Artemia salina: A microcrustacean to assess engineered nanoparticles toxicity.

The focus of this work was to investigate the toxicity of different metal nanoparticles (gold nanoparticles [AuNPs], silver nanoparticles [AgNPs], titanium dioxide nanoparticles [TiO2 NPs]) on brine shrimp Artemia salina. We investigated if nanoparticles could have an influence on hatching of cysts and on mortality of larvae. Larvae (also called nauplii) and cysts were exposed to NPs for 24 hr in artificial seawater on microplates. At the end of the test, we assessed the endpoint (immobility/death) for the larvae by a stereomicroscope. Nauplii that appeared completely motionless, were counted as dead, and the percentages of mortality were calculated for each treatment. Instead for the cysts, the percentages of not-hatched nauplii for each concentration considered were calculated by counting the number of whole cysts. Currently, nanoparticles toxicity has been investigated in several research; in our study we highlighted the nontoxicity of TiO2 NPs on A. salina nauplii as shown by low percentages of immobilization and on cysts because TiO2 NPs do not affect their hatching. Despite AuNPs exerted toxic effects on hatching, they did not affect larvae development as well as TiO2 NPs. Otherwise, AgNPs induced mortality of the larvae and inhibited cysts hatching.

Evaluation of dimethoate toxicity on fertilization and on embryonic development of Paracentrotus lividus (Lamarck, 1816).

Organophosphates are a large class of chemicals with anticholinesterase action insecticides. Dimethoate belongs to the class of organophosphates and it is used for agriculture purpose. Its main toxicological role in animals and humans is the inhibition of the activity of acetylcholinesterase. Although it is not considered genotoxic, carcinogenic and teratogen, there is evidence of increased pup mortality in developmental neurotoxicity studies. Since there is scant published literature about developmental toxicity, we investigated the adverse effects of dimethoate on fertilization and embryonic development in sea urchin (Paracentrotus lividus), a model organism widely used to assess the toxicity of contaminants on environmental matrices; so pesticide residues can be released into the environment, and could affect the health of organisms, including humans. Different solution of dimethoate (4 × 10-3, 4 × 10-4, 4 × 10-5, 4 × 10-6 and 4 × 10-7 g/10 ml) have been tested on spermatozoa of P. lividus to evaluate the fertilizing ability of them when we added egg cells untreated. We demonstrated that dimethoate does not interfere with fertilizing ability of spermatozoa but egg cells fertilized by treated spermatozoa showed alterations in the segmentation planes as asymmetric and/or asynchronous cell divisions.

Toxicity assessment of two-dimensional nanomaterials molybdenum disulfide in Gallus gallus domesticus.

Recently two-dimensional nanomaterials, such as graphene and molybdenum disulfide (MoS2), have received much attention as adsorbent materials for the effective removal of organic contaminants. MoS2 is attracting attention, not only for its chemical-physical properties, but also for its wide availability in nature as a constituent of molybdenite. The aim of this investigation was to assess the effects of different MoS2 concentrations (5 × 10-1, 5 × 10-2 and 5 × 10-3 mg/ml) on the embryonated eggs of Gallus gallus domesticus, according to Beck method. We evaluated the toxic effect of the MoS2 powder purchased at Sigma-Aldrich indicated as "received" and MoS2 powder treated via mechanical milling indicated as "ball mille". Subsequently, the embryos were sacrificed at different times of embryonic development (11th, 15th and 19th day after incubation) in order to evaluate their embryotoxic and teratogenic effects. The alterations of the embryonic development were studied by morphological and immunohistochemical analysis of the tissues. The results obtained have shown the toxicity of both powders of MoS2 with a high percentage of deaths and growth delays. Moreover, the immunohistochemical analysis performed on several tissue sections showed a strong positivity to the anti-metallothionein1 antibody only for the erythrocytes.

Ag/ZnO/PMMA Nanocomposites for Efficient Water Reuse.

This work attempts to produce photocatalytic surfaces for large-scale applications by depositing nanostructured coatings on polymeric substrates. ZnO/poly(methyl methacrylate) (PMMA) composites were prepared by low-temperature atomic layer deposition (ALD) of ZnO on PMMA substrates. In addition, to increase the photocatalytic and antibacterial activities of ZnO films, Ag nanoparticles were added on ZnO surfaces using plasma-enhanced ALD. The morphology, crystallinity, and chemical composition of the specimens were meticulously examined by scanning and transmission electron microscopies, energy-dispersive X-ray spectroscopy, and X-ray diffraction analyses. The noteworthy photocatalytic activity of the nanocomposites was proved by the degradation of the following organic pollutants in aqueous solution: methylene blue, paracetamol, and sodium lauryl sulfate. The antibacterial properties of the samples were tested using Escherichia coli as a model organism. Moreover, the possible toxic effects of the specimens were checked by biological tests. The present results unambiguously indicate the Ag/ZnO/PMMA nanocomposite as a powerful tool for an advanced wastewater treatment technology.

Toxicity assessment of nano-TiO2 in Apis mellifera L., 1758: histological and immunohistochemical assays.

The aim of this study was to evaluate the toxicity of titanium dioxide nanoparticles (TiO2 NPs) by short-term toxicity tests in Apis mellifera, considered an excellent bioindicator organism mainly due to its sensitivity. Bees have been exposed to several concentrations of TiO2 NPs (1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6 mg/10 ml) for 10 days. Morphostructural and histological assays were done on gut and honey sac. The research of exposure biomarkers like metallothioneins 1 (MT1) and Heat Shock Protein 70 (HSP70) was performed to verify if a detoxification mechanism has been activated in the exposed animals. No histological alteration on the epithelium of the gut and honey sac were observed in exposed samples. A significant positivity for anti-MT1 antibody was observed only in the honey sac cells. A weak positivity for HSP70 was observed in both structures analyzed. In several studies have shown the non-toxicity of TiO2 NPs on other model organisms, in our study, titanium dioxide nanoparticles was proven to be highly toxic at the highest concentration tested (100% of lethality to 1 × 10-3 mg/10 ml) and moderately toxic at lower concentrations. Honey bees proved to be excellent models for study of NPs toxicity and for monitoring environment.

Morphostructural and immunohistochemical study for evaluation of nano-TiO2 toxicity in Armadillo officinalis Duméril, 1816 (Crustacea, Isopoda, Oniscidea).

Invertebrates are precious organisms in order to study environmental pollution. In particular, they appear to be suitable as a bioindicator species for pioneer ecotoxicity studies on new xenobiotics such as nanoparticles. In fact, they are able to absorb nanomaterials scattered in the environment in different ways and it's known the compartmentalization of nano-sized contaminants in selected tissues and intracellular organelles. Titanium dioxide represents the most used nanoparticulate, destined to become probably ubiquitous in the environment. Recently, some research has been published on the toxic potential of nano-TiO2 in several animal species. Among all invertebrates, Oniscidean Isopods are the only taxon of Crustaceans that has become completely terrestrial, known as excellent bioindicators and bioaccumulators. They have a digestive gland, the hepatopancreas, which is the location of election for the accumulation of pollutants. For this reason, they are considered efficient animal models to ecological studies. For this study, we collected Armadillo officinalis from Natural Oriented Reserve of "Vendicari" (Sicily, Italy), to evaluate the toxicity of titanium dioxide (TiO2 ) on their hepatopancreas, after a short period of exposure. We conducted morphostructural and immunohistochemistry assays. The results suggested a great capacity of the species of bioaccumulation of nanoparticles in the hepatopancreas, where a strong positivity to the metallothioneins was highlighted. Our study confirms that Oniscidean Isopods, in particular Armadillo officinalis, proved to be an appropriate indicator of pollution in terrestrial ecosystems from nanoparticles.

Biomarkers of Exposure to Chemical Contamination in the Commercial Fish Species Lepidopus caudatus (Euphrasen, 1788): A Particular Focus on Plastic Additives.

In recent years, the Mediterranean Sea has become an accumulation zone for waste generated by the 22 countries bordering its shores. Although the effects of plastic litter on the marine environment and on organisms have recently been studied in other areas, further information is needed for the Mediterranean Sea and, in particular, about plastics additives inputs and interactions with the biota and the trophic network, such as phthalates and bisphenol A. Plastic material production, use and disposal contribute also to the release of heavy metals into the environment, such as mercury (Hg), often used during the production of chlorine, the primary ingredient in PVC, lead (Pb) and cadmium (Cd), which are used as stabilizers in PVC and leach out of products during use and disposal. Our research aims to evaluate phthalates, bisphenol A and heavy metals contamination in Lepidopus caudatus (Pisces, Trichiuridae), which could be considered as a potential sentinel species. For the evaluation of toxicological effects, we evaluated the expression of vitellogenin and metallothioneins 1. In all samples analyzed, we have not found microplastics in the gastrointestinal tract but chemical analysis revealed the presence of high content of phthalates, and in particular high quantities of DIDP, DEHP, bis-benzylester phthalate, bis-butyl ester phthalate and mono-N-butyl ester phthalate in different organs. Instead, trace elements detected in tissue revealed a trend of concentrations generally higher in liver and intestine than gill and muscle tissues. Immunohistochemical analysis for anti-metallothionein 1 antibody showed a strong positivity of liver cells, both in females and males. Analysis for the anti-vitellogenin antibody showed in females a strong positivity both in the liver cells and in the gonads, in male specimens was found to be always negative except for a specimen, in which it was highlighted a positivity in some areas of the liver and of the gonad.

Teratogenic effects of the neonicotinoid thiacloprid on chick embryos (Gallus gallus domesticus).

Thiacloprid is an insecticide belonging to the family of neonicotinoids, substances initially underestimated for their potential adverse effects, that they may manifest in the long term leading to an extensive use. The objective of this study was to evaluate the effect at increasing concentrations of thiacloprid on chick embryos development. The research was carried out on 75 fertile eggs of Gallus gallus domesticus. The eggs were opened after 10, 15 and 20 days of incubation and in treated embryos were observed developmental alterations, growth retardation, limbs defects and ectopia viscerale. The histological analysis showed hepatic steatosis and haemorrhages both in the liver and in the lungs. Moreover, the immunohistochemical analysis performed on the liver sections showed a strong positivity only for the erythrocytes to the anti-CYP1A antibody. Thiacloprid exposure increases the risks of teratogenic effects especially at the higher doses tested, therefore its use should be more controlled and limited. Since the literature on the topic is lacking, then the human health impacts resulting from neonicotinoids exposure is not yet fully understood, and, our data will be helpful to allow the assessment of an oral reference dose and health risk characterization.

Metallic Nano-Composite Toxicity Evaluation by Zebrafish Embryo Toxicity Test with Identification of Specific Exposure Biomarkers.

Nanomaterials (NM) have different shapes and can be composed of different materials such as carbon, silicon, and some metals like gold, silver, and titanium. They are used as fillers, catalysts, semiconductors, cosmetics, drug carriers in medicine, energy storage systems, and antifriction coatings. NM are the parent compounds of nanoparticles (NPs), which may be divided into two groups: fullerenes and engineered nanoparticles (ENPs). After crossing the cell membrane, NPs may be stored in vesicles, mitochondria, and additional organelles within epithelial cells. They may generate reactive oxygen species, oxidative stress, and cytotoxicity, as well as denaturation of proteins, apoptosis, and necrosis. Nowadays, new toxicological data are required to assess the potential exposure of the environment and human beings to pollutants. The aim of the present investigation is to evaluate the toxicity of the metallic nano-composite by the zebrafish embryo toxicity test (ZFET). The methods described here can be easily adapted to other nanocomposites or nanomaterials with some variations. © 2017 by John Wiley & Sons, Inc.

Morphostructural and immunohistochemical study on the role of metallothionein in the detoxification of heavy metals in Apis mellifera L., 1758.

Honey is a valuable food produced by bees from sugary substances that they gather in nature. The transformation the nectar into honey, by bees, is long and complex. Except for honey, where heavy metals are absent or are found only in traces, the bees and their products have always been considered excellent biomarkers of such contaminants. We have assumed that the absence of heavy metals in honey is due to the presence of a detoxification system in the digestive system of bees, which involves metallothioneins, proteins that have a role in the homeostatic control of essential and non-essential metals. We have placed the beehives in three different zones: industrial, urban and rural. Investigations were carried out with ICP-MS method for the detection of heavy metals in the guts of honey bees and honey. The metallothioneins have been identified by Immunohistochemical and Western-blotting analisys. The investigations have shown the presence of heavy metals only in bees guts but not in honey, while the presence of metallothionein has been highlighted only in epithelium of the honey sac, demonstrating the existence of an efficient system of detoxification of heavy metals.