Sandra Rainieri

Combined effects of microplastics and chemical contaminants on the organ toxicity of zebrafish (Danio rerio)

Abstract Microplastics contamination of the aquatic environment is considered a growing problem. The ingestion of microplastics has been documented for a variety of aquatic animals. Studies have shown the potential of microplastics to affect the bioavailability and uptake route of sorbed co‐contaminants of different nature in living organisms. Persistent organic pollutants and metals have been the co‐contaminants majorly investigated in this field. The combined effect of microplastics and sorbed co‐contaminants in aquatic organisms still needs to be properly understood. To address this, we have subjected zebrafish to four different feeds: A) untreated feed; B) feed supplemented with microplastics (LD‐PE 125–250 &mgr;m of diameter); C) feed supplemented with 2% microplastics to which a mixture of PCBs, BFRs, PFCs and methylmercury were sorbed; and D) feed supplemented with the mixture of contaminants only. After 3 weeks of exposure fish were dissected and liver, intestine, muscular tissue and brain were extracted. After visual observation, evaluation of differential gene expression of some selected biomarker genes in liver, intestine and brain were carried out. Additionally, quantification of perfluorinated compounds in liver, brain, muscular tissue and intestine of some selected samples were performed. The feed supplemented with microplastics with sorbed contaminants produced the most evident effects especially on the liver. The results indicate that microplastics alone does not produce relevant effects on zebrafish in the experimental conditions tested; on the contrary, the combined effect of microplastics and sorbed contaminants altered significantly their organs homeostasis in a greater manner than the contaminants alone. HighlightsMicroplastics did not produce effects on gene expression in zebrafish after 3 weeks.Microplastics and sorbed chemicals had a greater effect than chemicals alone.Effects were detected mainly in the liver.Perfluorinated compounds were detected mainly in the liver.Adverse effect of microplastics and sorbed chemicals should be considered in risk assessment.

Toxic effects of colloidal nanosilver in zebrafish embryos

A variety of consumer products containing silver nanoparticles (Ag NPs) are currently marketed. However, their safety for humans and for the environment has not yet been established and no standard method to assess their toxicity is currently available. The objective of this work was to develop an effective method to test Ag NP toxicity and to evaluate the effects of ion release and Ag NP size on a vertebrate model. To this aim, the zebrafish animal model was exposed to a solution of commercial nanosilver. While the exposure of embryos still surrounded by the chorion did not allow a definite estimation of the toxic effects exerted by the compound, the exposure for 48 h of 3‐day‐old zebrafish hatched embryos afforded a reliable evaluation of the effects of Ag NPs. The effects of the exposure were detected especially at molecular level; in fact, some selected genes expressed differentially after the exposure. The Ag NP toxic performance was due to the combined effect of Ag+ ion release and Ag NP size. However, the effect of NP size was particularly detectable at the lowest concentration of nanosilver tested (0.01 mg l–1) and depended on the solubilization media. The results obtained indicate that in vivo toxicity studies of nanosilver should be performed with ad hoc methods (in this case using hatched embryos) that might be different depending on the type of nanosilver. Moreover, the addition of this compound to commercial products should take into consideration the Ag NP solubilization media. Copyright

Zebrafish (Danio rerio) Larvae as a System to Test the Efficacy of Polysaccharides as Immunostimulants

The present study was carried out to examine the use of zebrafish (Danio rerio) as a preliminary screening model for testing the effect of potential immunostimulant substances on the innate immune system. β-Glucan, a polysaccharide used widely as an immunostimulant, was used as a representative molecule and tested on zebrafish embryos and larvae. The efficacy of the molecule was evaluated by determining the differential expression of some selected genes related to the immune system by RT-qPCR. Larvae from 72 hours post fertilization were found at the optimal developmental stage for assessing the expression of the selected genes. To verify if the β-glucan entered the larvae and therefore was responsible for the effects produced, the molecule was labeled fluorescently to check its localization by using microscopy. For estimating the effects of β-glucan on gene expression, zebrafish embryos and larvae were immersed in three different concentrations of β-glucan (50, 100, and 150 μg/mL) using five different exposure times. A stronger gene induction was observed when longer times of exposure and older larvae were used. The most evident effects of β-glucan were the overexpression of the genes TNFα, MPO, TRF, and LYZ. Moreover, slight changes in MPO expression were detected using a transgenic line of zebrafish (MPO::GFP), and a temporal increase in resistance against Vibrio anguillarum was found after β-glucan immersion. The assay used in this study permits the testing potential of immunostimulants in a simple and cost-effective way.

Occurrence and toxicity of musks and UV filters in the marine environment

Emerging chemical contaminants in the marine ecosystem represent a threat to the environment and also to human health due to insufficient knowledge about their toxicity and bioaccumulation in the food chain. Consequently, many of them are not regulated. In this review we focus on musks and organic UV filters. For both groups of compounds we describe occurrence in the marine environment, toxic effects identified so far and methods used to identify such effects. The final objective of this work is to identify gaps in the understanding of their toxicology.

Detection of exposure effects of mixtures of heavy polycyclic aromatic hydrocarbons in zebrafish embryos

In this study we evaluated the exposure effects of mixtures of five polycyclic aromatic hydrocarbons (PAHs); namely, benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene and chrysene on zebrafish embryos. Supplementation of the exposure media with 0.45% dimethyl sulfoxide and 50 ppm of Tween 20 could guarantee the solubilization and stabilization of the PAHs up to 24 h without affecting the embryos development. The exposure effects were tested by detecting the differential expression of a number of genes related to the aryl hydrocarbon receptor gene battery. Effects were detectable already after 6 h of exposure. After 24 h of exposure, all PAHs, except for benzo[a]anthracene, acted as potent inducers of the gene cyp1a1. Benzo[k]fluoranthene was the major inducer; the effect caused by the mixture at the lower concentration tested (1 ng ml−1) was dominated by its presence. However, in the mixture at the highest concentration tested (10 ng ml−1) it caused less induction and was not dominant. No significant bioaccumulation values were detected on embryos exposed to the PAHs tested in this study; however, the results obtained, indicated that PAHs undergo a very rapid metabolization inside the embryos, and that those biotransformation products yield changes on the expression of genes involved in the aryl hydrocarbon receptor pathway. Future work should focus on identification of the PAH metabolization products and on the effect of these metabolites on toxicity. Copyright

Toxic effects of perfluorinated compounds at human cellular level and on a model vertebrate

This work aims at deepening the understanding of the mode of action of some of the most prominent perfluorinated compounds (PFCs) by detecting in a realistic way their effects. To this end, after adjusting the exposure media taking into account the biological model employed and the physico-chemical properties of PFCs, we evaluated the toxic effects of PFOA, PFOS and PFNA in a human macrophage cell line (TLT cells) and in zebrafish embryos. We performed such evaluation on individual compounds and mixtures. Acute toxicity was greater for PFOS in zebrafish; however, it was greater for PFNA in TLT cells. PFNA was also the compound producing the greatest levels of oxidative stress, both in zebrafish and TLT cells. Additionally, in both biological systems, it showed a much stronger effect on mixtures in comparison to the others PFCs tested in this work. Mixture studies in zebrafish showed that acute toxicity depended on the concentration and that the mixture was far more toxic than the individual compounds. This study highlights the importance of studying PFCs in realistic conditions on various biological models.

Evaluation of MWNT toxic effects on daphnia and zebrafish embryos

Organisms of daphnia (Daphnia magna) and zebrafish (Danio rerio) embryos were exposed to a range of different concentrations of COOH-functionalized MWCNT suspended in an aqueous solution of Tween 20. Immobilization of daphnia and growth retardation, inhibition and malformation of zebrafish embryos were the endpoints tested after 24 and 48 hours. Immobilization of daphnia could be observed from 3 to 16 ppm and an increasing mortality of zebrafish embryo was detected at all the concentration tested. To identify more subtle toxic effects, we took advantage of the extensive information available on the zebrafish genome and monitored by RT-PCR the expression patterns of different zebrafish genes that could act as toxicity bio-markers. At some of the concentrations tested, changes in the expression profiles of the genes examined were detected. Our results suggest that MWCNT could potentially represent a risk to human health and environment, therefore a wider range of concentrations and further testing of this molecules should be carried out to define possible limitations in their use.

The influence of microplastics and halogenated contaminants in feed on toxicokinetics and gene expression in European seabass (Dicentrarchus labrax)

&NA; When microplastics pollute fish habitats, it may be ingested by fish, thereby contaminating fish with sorbed contaminants. The present study investigates how combinations of halogenated contaminants and microplastics associated with feed are able to alter toxicokinetics in European seabass and affect the fish. Microplastic particles (2%) were added to the feed either with sorbed contaminants or as a mixture of clean microplastics and chemical contaminants, and compared to feed containing contaminants without microplastics. For the contaminated microplastic diet, the accumulation of polychlorinated biphenyls (PCBs) and brominated flame retardants (BFRs) in fish was significantly higher, increasing up to 40 days of accumulation and then reversing to values comparable to the other diets at the end of accumulation. The significant gene expression results of liver (cyp1a, il1&bgr;, gst&agr;) after 40 days of exposure indicate that microplastics might indeed exacerbate the toxic effects (liver metabolism, immune system, oxidative stress) of some chemical contaminants sorbed to microplastics. Seabass quickly metabolised BDE99 to BDE47 by debromination, probably mediated by deiodinase enzymes, and unlike other contaminants, this metabolism was unaffected by the presence of microplastics. For the other PCBs and BFRs, the elimination coefficients were significantly lower in fish fed the diet with contaminants sorbed to microplastic compared to the other diets. The results indicate that microplastics affects liver detoxification and lipid distribution, both of which affect the concentration of contaminants. Graphical abstract Figure. No Caption available. HighlightsMicroplastics in feed influenced the toxicokinetics and affected the liver.kel of PCBs and BFRs lower for diet with contaminants sorbed to microplastics compared to other contaminated diets.The assimilation efficiencies lower for the contaminated diet with clean microplastics, likely due to higher eliminations.Seabass quickly metabolised PBDE99 to PBDE47 unaffected by the presence of microplastics.Significant gene expression of livers (cyp1a, il1&bgr;, gst&agr;) indicate that contaminants sorbed to microplastics increase the toxicity of some contaminants.

Lipid Nanoparticles: Delivery System for Bioactive Food Compounds

Food products containing bioactive ingredients with a health-promoting or disease-preventing effect represent an emerging market. The encapsulation of bioactive compounds can increase their stability, improve bioavailability, and mask undesirable taste. Nanoparticles exhibit some advantages over microparticles due to their size-dependent properties, but nano-scale systems need to undergo careful toxicological safety evaluation before they can be used for human consumption. Due to the hydrophobicity of most bioactive compounds and to the fact that the presence of digestible lipids often facilitates the absorption of bioactive compounds, great interest has developed in the major lipid-based nanocarriers. In this chapter we review the characteristics of solid lipid nanoparticles and nanostructure lipid carriers, paying special attention to bioavailability and toxicological aspects. We also provide some information about the application of nanotechnology in the food sector.

Overview on the safety of commercial products containing metallic nanoparticles in the food sector

The objective of this work was to highlight the importance of testing in a realistic way, metallic nanoparticles-based products, used in the food sector in order to provide representative data for a realistic and accurate risk assessment. To this end we give a brief overview of the current use and applications of metallic nanoparticles in the food industry, how the toxicological evaluations of nanoparticles in food should be performed and how nanoparticles-based products are regulated worldwide.This brief overview work provides a picture of the latest issues on the safety evaluation of metallic nanoparticles in the food sector and identifies also some major points that are important to take into consideration for future studies.