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The massive accumulation of plastics over the decades in the aquatic environment has led to the dispersion of plastic components in aquatic ecosystems, invading the food webs. Plastics fragmented into microplastics can be bioaccumulated by fishes via different exposure routes, causing several adverse effects. In the present study, the dose-dependent cytotoxicity of 8–10 μm polypropylene microplastics (PP-MPs), at concentrations of 1 mg/g (low dose) and 10 mg/g dry food (high dose), was evaluated in the liver and gill tissues of two fish species, the zebrafish (Danio rerio) and the freshwater perch (Perca fluviatilis). According to our results, the inclusion of PP-MPs in the feed of D. rerio and P. fluviatilis hampered the cellular function of the gills and hepatic cells by lipid peroxidation, DNA damage, protein ubiquitination, apoptosis, autophagy, and changes in metabolite concentration, providing evidence that the toxicity of PP-MPs is dose dependent. With regard to the individual assays tested in the present study, the biggest impact was observed in DNA damage, which exhibited a maximum increase of 18.34-fold in the liver of D. rerio. The sensitivity of the two fish species studied differed, while no clear tissue specificity in both fish species was observed. The metabolome of both tissues was altered in both treatments, while tryptophan and nicotinic acid exhibited the greatest decrease among all metabolites in all treatments in comparison to the control. The battery of biomarkers used in the present study as well as metabolomic changes could be suggested as early-warning signals for the assessment of the aquatic environment quality against MPs. In addition, our results contribute to the elucidation of the mechanism induced by nanomaterials on tissues of aquatic organisms, since comprehending the magnitude of their impact on aquatic ecosystems is of great importance.

The built up of microplastic (MPs) remains is shaping a new aquatic habitat and imposes the necessity for research of the effects that these relatively new pollutants exert on organisms, environment, and human health. The purpose of the present study was to verify if there is a particle-size dependence of fish response to MPs. Thus, we exposed two freshwater fish species, the zebrafish (Danio rerio) and perch (Perca fluviatilis) for 21 days to polyethylene microplastics (PE-MPs) sized 10-45 μm and 106-125 μm. Thereafter, in the liver and gills tissues, biochemical and molecular parameters and the metabolic profile were examined. Ex-vivo characterization by ATR-FTIR spectroscopy exhibited increased concentration of 10-45 μm PE-MPs in the liver of the two fish species while 106-125 μm PE-MPs mostly concentrated in fish gills. The penetration of PE-MPs to fish and the induced oxidative stress triggered changes in lipid peroxidation, DNA damage and ubiquitination and furthermore stimulated signal transduction pathways leading to autophagy and apoptosis. The smaller PE-MPs were more potent in inducing alterations to all the latter parameters measured than the larger ones. Tissue response in both fish seems to depend on the parameter measured and does not seem to follow a specific pattern. Our results showed that there is no clear sensitivity of one fish species versus the other, against both sizes of PE-MPs they were exposed. In perch the metabolic changes in gills were distinct to the ones observed in liver, following a size dependent pattern, indicating that stress conditions are generated through different mechanisms. All the parameters employed can be suggested further as biomarkers in biomonitoring studies against PE-MPs.

The goal of the present study was to examine the effects of ZnO NPs and CuO NPs on Cornu aspersum land snail, enlightening their cytotoxic profile. ZnO NPs and CuO NPs were synthesized and thoroughly characterized. Α series of concentrations of either ZnO NPs or CuO NPs were administered in the feed of snails for 20 days. Thereafter, neutral red retention assay was conducted, in order to estimate NRRT50 values. Subsequently, snails were fed with NPs concentrations slightly lower than the concentrations that were corresponding to the NRRT50 values, i.e. 3 mg·L−1 ZnO NPs and 6 mg·L−1 CuO NPs, for 1, 5, 10 and 20 days. Both NPs agglomerates were detected in hemocytes by Transmission Electron Microscopy. Moreover, both effectors resulted to toxicity in the snails' hemocytes. The latter was shown by changes in the NRRT50 values, increased reactive oxygen species (ROS) production, lipid peroxidation, DNA integrity loss, protein carbonyl content, ubiquitin conjugates and cleaved caspases conjugates levels compared to the untreated animals. Although ZnO NPs exhibited higher toxicity, as indicated by the NRRT50 values, both NPs affected similarly a wide range of the cellular parameters mentioned above. The latter parameters could constitute sensitive biomarkers in biomonitoring studies of terrestrial environment against nanoparticles.

In the present study the effects of sublethal concentrations of polystyrene microplastics (PS-MPs) on zebrafish were evaluated at multiple levels, related to fish activity and oxidative stress, metabolic changes and contraction parameters in the heart tissue. Zebrafish were fed for 21 days food enriched with PS-MPs (particle sizes 3–12 µm) and a battery of stress indices like DNA damage, lipid peroxidation, autophagy, ubiquitin levels, caspases activation, metabolite adjustments, frequency and force of ventricular contraction were measured in fish heart, parallel to fish swimming velocity. In particular, exposure to PS-MPs caused significant decrease in heart function and swimming competence, while enhanced levels of oxidative stress indices and metabolic adjustments were observed in the heart of challenged species. Among stress indices, DNA damage was more vulnerable to the effect of PS-MPs. Our results provide evidence on the multiplicity of the PS-MPs effects on cellular function, physiology and metabolic pathways and heart rate of adult fish and subsequent effects on fish activity and fish fitness thus enlightening MPs characterization as a potent environmental pollutant.

Titanium dioxide nanoparticles (TiO

Among pollutants, nanoparticles (NPs) consist a potential environmental hazard, as they could possibly harm the aquatic and terrestrial ecosystems while having unpredictable repercussions on human health. Since monitoring the impact of NPs on aquatic and terrestrial life is challenging, due to the differential sensitivities of organisms to a given nanomaterial, the present study examines magnetite nanoparticles' mediated toxicity in different animal models, representing distinctive environments (terrestrial and aquatic). Oxidative, proteolytic and genotoxic effects were evaluated on the hemocytes of the snail Cornu aspersum; in addition to those, apoptotic effects were measured in gills and liver of the zebrafish Danio rerio, and the prussian carp Carassius gibelio. All biochemical parameters studied increased significantly in animals after 8 days exposure to NPs. Inter-species and inter-tissues differences in responses were evident. Our results suggest a common toxicity response mechanism functioning in the tissues of the three animals studied that is triggered by magnetite NPs. The simultaneous use of these parameters could be established after further investigation as a reliable multi-parameter approach for biomonitoring of terrestrial and aquatic ecosystems against magnetite nanoparticles. Additionally, the results of our study could contribute to the design of studies for the production and rational utilization of nanoparticles.

Considering that the extensive biomedical, pharmaceutics, cosmetic and other industrial applications of biomaterials (BMs) is of great concern nowadays, regarding their environmental risk, the present study aimed to investigate the effects of four BMs, poly(e-caprolactone) (PCL), poly(butylene succinate) (PBSu), chitosan (CS) and modified chitosan (succinic acid grafted chitosan) (CS-Suc) in the form of microplastics (particle sizes less than 1 mm) on biochemical parameters of snails Cornu aspersum hemocytes. Due to the absence of knowledge about the environmentally relevant concentrations of BMs, snails were initially treated through their food with a wide range of nominal concentrations of each BM to define the half maximal effective concentration (NRRT50), according to the destabilization degree of hemocytes’ lysosomal membranes (by mean of neutral red retention time/NRRT assay). Thereafter, snails were treated with each BM, at concentrations lower than the estimated NRRT50 values in all cases, for periods up to 15 days. After the end of the exposure period, a battery of stress indices were measured in hemocytes of challenged snails. According to the results, all parameters tested in BMs-treated snails statistically differed from those measured in BMs-free snails, thus indicating the pro-oxidant potential of BMs, as well as their ability to affect animals’ physiology. The most considerable effect in most cases seems to be caused by modified chitosan and PCL, while chitosan appears to be the least toxic. A common response mechanism of snails’ blood cells against the 4 BMs used in the present study was shown. After exposure to each of the studied BMs a significant augmentation in protein carbonyls, MDA equivalents and DNA damage, while a significant reduction in NRRT values was determined in the snails hemocytes, in relation to the unexposed animals. From the biochemical parameters examined, MDA equivalents and DNA damage seem to be more susceptible than the other parameters studied, to respond to BMs effect, with MDA to react with more sensitivity to PCL and CS, while DNA damage to CS-Suc and PBSu. Our results could suggest the simultaneous use of the latter biomarkers in biomonitoring studies of terrestrial ecosystems against the specific BMs.

Nanoparticles (NPs), due to their increased application and production, are being released into the environment with unpredictable impact on the physiology of marine organisms, as well as on entire ecosystems and upcoming effects on human health. The aim of the present study was to evaluate and compare the oxidative responses of the mussel Mytilus galloprovincialis after exposure to iron oxide NPs and to iron oxide NPs incorporated into zeolite for 1, 3 and 7 days. Our results showed that both effectors induced changes on animal physiology by causing oxidative stress in hemocytes of exposed mussels compared to control animals. This was shown by the significant increase in reactive oxygen species (ROS) production, protein carbonylation, lipid peroxidation, ubiquitin conjugates and DNA damage. In addition an increase in prooxidant levels as measured by the prooxidant-antioxidant balance (PAB) assay was observed in exposed mussels' hemolymph. The results show that ROS, DNA damage, protein and lipid oxidation, ubiquitin conjugates and PAB could constitute, after further investigation, reliable biomarkers for the evaluation of pollution or other environmental stressors. In addition, more studies are needed in order to ensure the safety of these NPs on various biomedical applications, since it is critical to design NPs that they meet the demands of application without causing cellular toxicity.

The use of manufactured nanoparticles (NPs) is spreading rapidly across technology and medicine fields, posing concerns about their consequence on ecosystems and human health. The present study aims to assess the biological responses triggered by iron oxide NPs (IONPs) and iron oxide NPs incorporated into zeolite (IONPZ) in relation to oxidative stress on the land snail Helix aspersa in order to investigate its use as a biomarker for terrestrial environments. Morphology and structure of both NPs were characterized. Snail food was supplemented with a range of concentrations of IONPs and IONPZ and values of the hemocyte lysosomal membranes' destabilization by 50% were estimated by the neutral red retention (NRRT50) assay. Subsequently, snails were fed with NPs concentrations equal to half of the NRRT50 values, 0.05 mg L-1 for IONPs and 1 mg L-1 for IONPZ, for 1, 5, 10 and 20 days. Both effectors induced oxidative stress in snails' hemocytes compared to untreated animals. The latter was detected by NRRT changes, reactive oxygen species (ROS) production, lipid peroxidation estimation, DNA integrity loss, measurement of protein carbonyl content by an enzyme-linked immunoabsorbent assay (ELISA), determination of ubiquitin conjugates and cleaved caspases conjugates levels. The results showed that the simultaneous use of the parameters tested could constitute possible reliable biomarkers for the evaluation of NPs toxicity. However, more research is required in order to enlighten the disposal and toxic impact of iron oxide NPs on the environment to ensure their safe use in the future.

Georgia Kastrinaki,1,* Christos Samsouris,2,* Efstratios K Kosmidis,3 Eleni Papaioannou,1 Athanasios G Konstandopoulos,1,4 George Theophilidis2 1Aerosol and Particle Technology Laboratory (APTL), CERTH/CPERI, Thessaloniki, Greece; 2Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece; 3Laboratory of Physiology, Department of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece; 4Department of Chemical Engineering, Aristotle University of Thessaloniki, Thessaloniki, Greece *These authors contributed equally tothis work Abstract: The axonal translocation of two commonly used nanoparticles in medicine, namely CeO2 and SiO2, is investigated. The study was conducted on frog sciatic nerve fibers in an exvivo preparation. Nanoparticles were applied at the proximal end of the excised nerve. A nerve stimulation protocol was followed for over 35 hours. Nerve vitality curve comparison between control and exposed nerves showed that CeO2 has no neurotoxic effect at the concentrations tested. After exposure, specimens were fixed and then screen scanned every 1mm along their length for nanoparticle presence by means of Fourier transform infrared microscopy. We demonstrated that both nanoparticles translocate within the nerve by formation of narrow bands in the Fourier transform infrared spectrum. For the CeO2, we also demonstrated that the translocation depends on both axonal integrity and electrical activity. The speed of translocation for the two species was estimated in the range of 0.45–0.58 mm/h, close to slow axonal transportation rate. Transmission electron microscopy provided direct evidence for the presence of SiO2 in the treated nerves. Keywords: CeO2, SiO2, FTIR, nanoparticles, ex vivo electrophysiology, frog sciatic nerve, translocation