Genotoxicity and metabolic changes induced via ingestion of virgin and UV-aged polyethylene microplastics by the freshwater fish Perca fluviatilis.

The present study aims to compare and assess the toxicity induced by aged (irradiated with ultraviolet radiation for 120 days) polyethylene microplastics (PE-MPs) in comparison to virgin (non-irradiated) ones, after feeding the freshwater fish Perca fluviatilis. To this end, MPs mediated genotoxicity was assessed by the investigation of micronucleus nuclear abnormalities frequency in fish blood, and the degree of DNA damage in the liver and muscle tissues, while metabolic alterations were also recorded in both tissues. Results showed that both virgin and aged PE-MPs induced signaling pathways leading to DNA damage and nuclear abnormalities, as well as metabolites changes in all tissues studied. Metabolic changes revealed that the metabolism of nucleic acids, energy, amino acids, and neurotransmitters was more disrupted in the liver and by aged PE-MPs compared to muscles. Fish fed with aged PE-MPs exhibited greater DNA damage, while blood cells of fish fed with virgin PE-MPs seemed to be more vulnerable to nuclear abnormalities in relation to those fed with aged PE-MPs. Moreover, aged PE-MPs induced more acute overall effects on the metabolic profiles of fish tissues, and initiated stronger stress responses, inflammation, and cellular damages in fish tissues in relation to virgin ones. Characterization of both virgin and aged MPs revealed that the latter exhibited lower crystallinity and melting point, more irregular shapes and higher moiety of oxygen and carbonyl groups, which could be attributed for their observed higher toxicity. The research outcomes provide significant insights for advancing toxicological investigations in this field.

Dose-Dependent Cytotoxicity of Polypropylene Microplastics (PP-MPs) in Two Freshwater Fishes.

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.

Do poly(lactic acid) microplastics instigate a threat? A perception for their dynamic towards environmental pollution and toxicity.

Fears concerning microplastics (MPs) environmental fate and persistence are progressively expanding on a global basis, with the emphasis given to manufacturing bioplastics for substituting petro-derived plastics extensively growing. Among them, poly(lactic acid) (PLA) holds a pioneering role towards the replacement of conventional polymeric materials, owing to its multifunctional properties, enclosing superior mechanical properties, low cost, renewability, great biocompatibility, transparency, and thermoplasticity launching many fields of application. Due to the wide applicability of PLA in several sectors of everyday life, its waste to be released into the environment is expected to follow a growing tendency during the upcoming years. Even though PLA is a biodegradable polyester, it actually degrades under specific composting environments, including a rich oxygen environment with high temperatures (58-80 °C), high humidity (>60% moisture) as well as the presence of micro-organisms (thermophilic bacteria). Additionally, in various studies it has been implied that PLA displays slower degradation performance when found in blends with other conventional polymers, underlining the unspecified effects on PLA degradation profile, keeping thus the information about PLA degradation from a blur standpoint. Therefore, a deepened understanding of the fate and dynamic effects of PLA MPs is of primary importance. Nevertheless, the current examination of the effects of PLA MPs in terms of sorption capacities and toxicity is so far limited and broadly unexplored since the current scientific emphasis has been merely centered on the conventional MPs' behavior. In this light, the present review provides an inclusive overview of the ongoing research of poly(lactic acid) in the framework of microplastics' pollution, while the future trends and missing points in this context are highlighted.

Differentiation in the expression of toxic effects of polyethylene-microplastics on two freshwater fish species: Size matters.

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.

DNA barcoding identification of Greek freshwater fishes.

Biodiversity is a key factor for the functioning and efficiency of an ecosystem. Greece, though covering a relatively small surface area, hosts a great deal of species diversity. This is especially true for freshwater fishes. In recent years, the traditional methods of species identification have been supplemented by the use of molecular markers. The present study therefore aims to extensively produce DNA barcodes for Greek freshwater fish species and investigate thoroughly if the presently accepted species classification is in agreement with molecular data. A 624-bases long fragment of the COI gene was sequenced, from 406 freshwater fish specimens belonging to 24 genera and originating from 18 lake and river sites. These sequences were used along with 596 sequences from the same genera, recovered from BOLD, for the construction of phylogenetic trees and the estimation of genetic distances between individuals. In total, 1002 sequences belonging to 72 species were analyzed. The method was found to be effective for 55 of 72 studied species. 17 closely related species with low interspecific genetic distances were observed, for which further study is proposed. It should also be noted that, in four cases, cryptic diversity was observed, where groups originally identified as one species exhibited genetic distance great enough to be separated into discrete species. Region specific haplotypes were also detected within populations of 14 species, giving the possibility to identify even the geographic origin of a species. Our findings are discussed in the light of the rich history of the Balkan peninsula and provide a significant steppingstone for the further study of Greek and European freshwater fish biodiversity.

Toxicity and Functional Tissue Responses of Two Freshwater Fish after Exposure to Polystyrene Microplastics.

Microplastics (MPs)' ingestion has been demonstrated in several aquatic organisms. This process may facilitate the hydrophobic waterborne pollutants or chemical additives transfer to biota. In the present study the suitability of a battery of biomarkers on oxidative stress, physiology, tissue function and metabolic profile was investigated for the early detection of adverse effects of 21-day exposure to polystyrene microplastics (PS-MPs, sized 5-12 µm) in the liver and gills of zebrafish Danio rerio and perch, Perca fluviatilis, both of which are freshwater fish species. An optical volume map representation of the zebrafish gill by Raman spectroscopy depicted 5 µm diameter PS-MP dispersed in the gill tissue. Concentrations of PS-MPs close to the EC50 of each fish affected fish physiology in all tissues studied. Increased levels of biomarkers of oxidative damage in exposed fish in relation to controls were observed, as well as activation of apoptosis and autophagy processes. Malondialdehyde (MDA), protein carbonyls and DNA damage responses differed with regard to the sensitivity of each tissue of each fish. In the toxicity cascade gills seemed to be more liable to respond to PS-MPs than liver for the majority of the parameters measured. DNA damage was the most susceptible biomarker exhibiting greater response in the liver of both species. The interaction between MPs and cellular components provoked metabolic alterations in the tissues studied, affecting mainly amino acids, nitrogen and energy metabolism. Toxicity was species and tissue specific, with specific biomarkers responding differently in gills and in liver. The fish species that seemed to be more susceptible to MPs at the conditions studied, was P. fluviatilis compared to D. rerio. The current findings add to a holistic approach for the identification of small sized PS-MPs' biological effects in fish, thus aiming to provide evidence regarding PS-MPs' environmental impact on wild fish populations and food safety and adequacy.

Adverse effects polystyrene microplastics exert on zebrafish heart - Molecular to individual level.

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.

Common mechanisms activated in the tissues of aquatic and terrestrial animal models after TiO2 nanoparticles exposure.

Titanium dioxide nanoparticles (TiO2-NPs) are among the most popular manufactured and widely used nanoparticles. They are released into the environment, affecting terrestrial and aquatic ecosystems, with unexpected consequences to organisms and human health. The present study investigates the mediated toxicity imposed to the freshwater fish species, zebrafish (Danio rerio) and the prussian carp (Carassius gibelio), and to the terrestrial land snail Cornu aspersum, after their exposure to sublethal concentrations of TiO2-NPs. Oxidative, proteolytic, genotoxic and apoptotic parameters in fish liver and gills, as well as on snail hemocytes were studied and the swimming performance was estimated in order to (a) estimate and suggest the most susceptible animal, and (b) propose a common battery of biomarkers as the most suitable indicator for biomonitoring studies against TiO2-NPs. Our in vivo experiments demonstrated that NPs induced detrimental effects on animal physiology and swimming behavior, while no general pattern was observed in species and tissues responsiveness. Generally, TiO2-NPs seemed to activate a group of molecules that are common for aquatic as well as terrestrial animals, implying the existence of a conserved mechanism. It seems that after exposure to TiO2-NPs, a common mechanism is activated that involves the stimulation of immune system with the production of ROS, damage of lysosomal membrane, protein carbonylation, lipid peroxidation, DNA damage, following proteolysis by ubiquitin and finally apoptosis. Thus, the simultaneous use of the latter biomarkers could be suggested as a reliable multi parameter approach for biomonitoring of aquatic and terrestrial ecosystems against TiO2-NPs.

Magnetite nanoparticles effects on adverse responses of aquatic and terrestrial animal models.

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.

Biochemical and molecular responses of cyprinids in two Mediterranean lacustrine ecosystems: Opportunities for ecological assessment and biomonitoring.

Lacustrine ecosystems have been altered by accelerating pollution, excessive nutrient and organic load, water abstraction, and are susceptible to climate change. Hence, suggesting sensitive and reliable biomarkers for early assessments of their status is of urgent need. In this study, two freshwater commercial fish species, Cyprinus carpio (carp) and Carassius gibelio (prussian carp) from two lakes (i.e. Koronia and Volvi, Northern Greece) with different anthropogenic pressures were used and a battery of biochemical and molecular biomarkers related to stress response were analyzed in fish gills and liver. In parallel, water physicochemical parameters (T, DO, pH, conductivity, salinity), BOD5 and nutrient (N-NO3, N-NO2, N-NH4, P-PO4) concentrations were measured. Results showed that Lake Koronia had higher conductivity and salinity values and N-NO2 concentrations. Levels of Heat Shock Response (HSR), MAPK phosphorylation, protein carbonylation, lipid peroxidation products, Bax/Bcl-2 ratio, ubiquitination and caspases were increased in gills and liver of both fish species sampled from Lake Koronia in relation to those of Lake Volvi. Likewise, liver lipid content was increased in both fish species sampled from Lake Koronia compared to those sampled from Lake Volvi. The results indicate and reflect the higher environmental degradation that prevails in Lake Koronia ecosystem in comparison to that of Lake Volvi. The fish species studied showed different susceptibility depending on the biomarkers examined. In addition, our results from both examined species provide insight into the mechanisms involved in acclimatization to stressful environments and support the role of the studied biomarkers as sensitive and reliable tools for ecological assessments of lake ecosystems in biomonitoring studies.

DNA barcoding analysis of fish species diversity in four north Greek lakes.

MATERIALS AND METHODS: The present study is the first to apply DNA barcoding on identifying 37 freshwater fish species from the rich Balkan ichthyofauna. RESULTS: The results are highly successful since in most cases barcodes cluster according to species, in agreement with morphological taxonomic studies. This is also evident based on mean conspecific and congeneric Kimura two-parameter distance values. The 5.6-fold difference between these values is lower than previous barcoding studies, possibly due to the restricted samplings and the recent taxonomy reevaluation for several species. A number of species were identified, where future work is needed: For the species Scardinius erythrophthalmus, Perca fluviatilis, and Rutilus rutilus, the divergence values found among conspecific populations could warrant their placement into different species; for Barbus and Rhodeus populations, the reported interspecific distances found were lower than expected; and for Cobitis species, the application of barcoding seems problematic, due to their complicated reproduction. CONCLUSION: The extension of this work to other Greek or even Balkan freshwater systems should clarify the situation.

Assessing ecological water quality with macroinvertebrates and fish: a case study from a small Mediterranean river.

Biological elements, such as benthic macroinvertebrates and fish, have been used in assessing the ecological quality of rivers according to the requirements of the Water Framework Directive. However, the concurrent use of multiple organism groups provides a broader perspective for such evaluations, since each biological element may respond differently to certain environmental variables. In the present study, we assessed the ecological quality of a Greek river (RM4 type), during autumn 2003 and spring 2004 at 10 sites, with benthic macroinvertebrates and fish. Hydromorphological and physicochemical parameters, habitat structure, and riparian vegetation were also considered. Pollution sensitive macroinvertebrate taxa were more abundant at headwaters, which had good/excellent water quality according to the Hellenic Evaluation System (HES). The main river reaches possessed moderate water quality, while downstream sites were mainly characterised as having bad or poor water quality, dominated by pollution-tolerant macroinvertebrate taxa. Macroinvertebrates related strongly to local stressors as chemical degradation (ordination analysis CCA) and riparian quality impairment (bivariate analysis) while fish did not. Fish were absent from the severely impacted lower river reaches. Furthermore, external pathological signs were observed in fish caught at certain sites. A combined use of both macroinvertebrates and fish in biomonitoring programs is proposed for providing a safer assessment of local and regional habitat impairment.

Seasonal variation of fish abundance and biomass in gillnet catches of an East Mediterranean lake: Lake Doirani.

The seasonal variation of fish species composition and abundance in gillnet catches (14-90 mm knot-to-knot) from the Greek part of the transboundary Lake Doirani was studied during the period 2006-2007. A total of 8,419 specimens weighing 182.3 kg and belonging to 9 species were caught. Catch composition differed with season. Thus, Rhodeus meridionalis dominated in terms of NPUE the spring, Perca fluviatilis the summer and Albumus macedonicus the autumn and winter catches. Cyprinids were generally the most abundant, with the cyprinids:percids biomass ratio ranging from 1.7 in summer to 14.8 in winter, supporting the eutrophic character of the lake. Richness and Shannon-Wiener diversity and evenness indices differed seasonally (ANOVA; p < 0.05). The abundance-biomass comparative (ABC) curves showed that fish communities were dominated by one or a few opportunistic species (e.g. Rhodeus meridionalis, Albumus macedonicus), which while dominated in number did not dominate in biomass, being small bodied.