Plastic microfibers (MF) represent the major source of MF found in the environment, the majority made of polyester (PES). Marine bivalves, suspension feeders widespread in coastal areas subjected to ...higher anthropogenic input, can accumulate MF from the water column in their tissues. This raised some concern about their possible impact on bivalve health and potential transfer along the food chain. In this work, the effects of PES-MF on the mussel Mytilus galloprovincialis were investigated, utilizing MF obtained by cryo-milling of a fleece cover. Fiber characterization indicated the polymer composition as polyethylene terephthalate (PET); the size distribution was in a length range resembling that of MF released from textile washing, and including those that can be ingested by mussels. MF were first screened for short-term in vitro immune responses in mussel hemocytes. The effects of in vivo exposure (96 h, 10 and 100 μg/L, corresponding to about 150 and 1500 MF/mussel/L, respectively), were then evaluated. Data are presented on hemolymph immune biomarkers (Reactive Oxygen Species and nitric oxide production, lysozyme activity), and on antioxidant biomarkers (catalase and glutathione S-transferase) and histopathology in gills and digestive gland. Tissue MF accumulation was also evaluated.
MF exposure stimulated extracellular immune responses both in vitro and in vivo, indicating induction of immune/inflammatory processes. In both tissues, stimulation of antioxidant enzyme activities, suggesting oxidative stress conditions, and histopathological changes were observed, with stronger effects often observed at lower concentration. Although mussel retained a very small fraction of MF, their accumulation was higher in the digestive gland than in gills, and in both tissues of mussels exposed to the lowest concentration. Selective accumulation of shorter MF was also observed, particularly in gills. Overall, the results demonstrate that at environmental exposure levels, PET-MF have a significant impact on mussel physiology, affecting multiple processes in different tissues.
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•Effects of heterogeneous PES MF on the mussel M. galloprovincialis•In vitro MF stimulate immune responses in the hemocytes.•In vivo exposure induced tissue inflammation and oxidative stress.•Histopathological changes in gills and digestive gland•Small but selective accumulation of shorter MFs
When a new approach in microscopy is introduced, broad interest is attracted only when the sample preparation procedure is elaborated and the results compared with the outcome of the existing ...methods. In the work presented here we tested different preparation procedures for focused ion beam (FIB) milling and scanning electron microscopy (SEM) of biological samples. The digestive gland epithelium of a terrestrial crustacean was prepared in a parallel for FIB/SEM and transmission electron microscope (TEM). All samples were aldehyde-fixed but followed by different further preparation steps. The results demonstrate that the FIB/SEM samples prepared for conventional scanning electron microscopy (dried) is suited for characterization of those intracellular morphological features, which have membranous/lamellar appearance and structures with composition of different density as the rest of the cell. The FIB/SEM of dried samples did not allow unambiguous recognition of cellular organelles. However, cellular organelles can be recognized by FIB/SEM when samples are embedded in plastic as for TEM and imaged by backscattered electrons. The best results in terms of topographical contrast on FIB milled dried samples were obtained when samples were aldehyde-fixed and conductively stained with the OTOTO method (osmium tetroxide/thiocarbohydrazide/osmium tetroxide/thiocarbohydrazide/osmium tetroxide). In the work presented here we provide evidence that FIB/SEM enables both, detailed recognition of cell ultrastructure, when samples are plastic embedded as for TEM or investigation of sample surface morphology and subcellular composition, when samples are dried as for conventional SEM.
Extensive production and wide application of TiO₂ nanoparticles has stimulated research on its potential biological effects on different groups of organisms but the interaction of TiO₂ nanoparticles ...with higher plants remains poorly understood. We have studied the effect of TiO₂ nanoparticles on Allium cepa using a modification of the conventional Allium test with nanoparticles suspended in distilled water as opposed to growth medium. Nanoparticulate TiO₂ was found to have low toxic potential and the mitotic index was among the most sensitive measures of the effect of nano-TiO₂. We conclude that modified Allium test is suitable to provide comparative data on the biological potential of a variety of nanoparticles and could be used in a tiered approach to nanotoxicity testing.
► Tetrahymena thermophila was exposed to TiO2 particles at sub-toxic concentrations. ► Exposure to TiO2 particles resulted in changed cell membrane in fatty acid profile resulting in increased ...membrane rigidity. ► Altered cell membrane composition is not accompanied by lipid peroxidation or ROS elevation. ► Particles were detected in food vacuoles where filling and subsequent release was followed.
We provide experimental evidence that changes in the membrane fatty acid profile of Tetrahymena thermophila incubated with nano- or bulk TiO2 particle are not accompanied by ROS generation or lipid peroxidation. Consequently these changes are interpreted as acclimation to unfavorable conditions and not as toxic effects. T. thermophila cells were exposed to TiO2 particles at different concentrations for 24h at 32°C. Treatment of cultures with nano- and bulk TiO2 particles resulted in changes of membrane fatty acid profile, indicating increased membrane rigidity, but no lipid peroxidation or ROS generation was detected. There were no differences in membrane composition when T. thermophila was exposed to nanosized or bulk-TiO2 particles. We also observed reversible filling of food vacuoles, but this was different in case of nano- or bulk TiO2 exposure. Our results suggest that interactions of particles and cell membranes are independent of oxidative stress.
Use of a modified Allium test with nanoTiO2 KLANCNIK, K; DROBNE, D; VALANT, J ...
Ecotoxicology and environmental safety,
2011, 2011-Jan, 2011-01-00, 20110101, Volume:
74, Issue:
1
Journal Article
Peer reviewed
Extensive production and wide application of TiO(2) nanoparticles has stimulated research on its potential biological effects on different groups of organisms but the interaction of TiO(2) ...nanoparticles with higher plants remains poorly understood. We have studied the effect of TiO(2) nanoparticles on Allium cepa using a modification of the conventional Allium test with nanoparticles suspended in distilled water as opposed to growth medium. Nanoparticulate TiO(2) was found to have low toxic potential and the mitotic index was among the most sensitive measures of the effect of nano-TiO(2). We conclude that modified Allium test is suitable to provide comparative data on the biological potential of a variety of nanoparticles and could be used in a tiered approach to nanotoxicity testing.
The analysis of biological samples in frozen-hydrated state with micro-PIXE technique at Jozef Stefan Institute (JSI) nuclear microprobe has matured to a point that enables us to measure and examine ...frozen tissue samples routinely as a standard research method. Cryotome-cut slice of frozen-hydrated biological sample is mounted between two thin foils and positioned on the sample holder. The temperature of the cold stage in the measuring chamber is kept below 130K throughout the insertion of the samples and the proton beam exposure. Matrix composition of frozen-hydrated tissue is consisted mostly of ice. Sample deterioration during proton beam exposure is monitored during the experiment, as both Elastic Backscattering Spectrometry (EBS) and Scanning Transmission Ion Microscopy (STIM) in on-off axis geometry are recorded together with the events in two PIXE detectors and backscattered ions from the chopper in a single list-mode file. The aim of this experiment was to determine differences and similarities between two kinds of biological sample preparation techniques for micro-PIXE analysis, namely freeze-drying and frozen-hydrated sample preparation in order to evaluate the improvements in the elemental localisation of the latter technique if any. In the presented work, a standard micro-PIXE configuration for tissue mapping at JSI was used with five detection systems operating in parallel, with proton beam cross section of 1.01.0 mu m2 and a beam current of 100pA. The comparison of the resulting elemental distributions measured at the biological tissue prepared in the frozen-hydrated and in the freeze-dried state revealed differences in elemental distribution of particular elements at the cellular level due to the morphology alteration in particular tissue compartments induced either by water removal in the lyophilisation process or by unsatisfactory preparation of samples for cutting and mounting during the shock-freezing phase of sample preparation.
Terrestrial isopods are suitable invertebrates for testing the relative toxicities of chemicals present in the terrestrial environment. Terrestrial isopods respond in numerous ways to elevated ...concentrations of chemicals in their food, but only a few of these responses can be used as toxicological endpoints. The most suitable are changes in reproduction, food consumption, moult cycle duration, and structure of the digestive glands. These responses are able to provide accurate indications of sublethal toxicity. Toxicity tests with terrestrial isopods could be much more reliable through the use of positive controls. A positive control with a reference toxicant could also be supplemented by a reference endpoint. The most suitable reference endpoint is change of food consumption rate. Toxicity testing with terrestrial isopods is a very promising method for fast, routine, and inexpensive laboratory determination of the relative toxicities of chemicals in the terrestrial environment.
Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO
2
nanoparticles (TiO
2
-NPs) and their bulk counterparts ...(bulk-TiO
2
). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO
2
-NPs or bulk-TiO
2
particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO
2
particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO
2
-NPs concentration differed significantly from the response to higher TiO
2
-NPs concentration and both bulk-TiO
2
concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO
2
; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO
2
-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.
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