Biological Uptake, Distribution, and Depuration of Radio-Labeled Graphene in Adult Zebrafish: Effects of Graphene Size and Natural Organic Matter.
ABSTRACT: The exciting commercial application potential of graphene materials may inevitably lead to their increasing release into the environment where they may pose ecological risks. This study focused on using carbon-14-labeled few-layer graphene (FLG) to determine whether the size of graphene plays a role in its uptake, depuration, and biodistribution in adult zebrafish. After 48 h exposure to larger FLG (L-FLG) at 250 ?g/L, the amount of graphene in the organism was close to 48 mg/kg fish dry mass, which was more than 170-fold greater than the body burden of those exposed to the same concentration of smaller FLG (S-FLG). The amount of uptake for both L-FLG and S-FLG increased by a factor of 2.5 and 16, respectively, when natural organic matter (NOM) was added in the exposure suspension. While the L-FLG mainly accumulated in the gut of adult zebrafish, the S-FLG was found in both the gut and liver after exposure with or without NOM. Strikingly, the S-FLG was able to pass through the intestinal wall and enter the intestinal epithelial cells and blood. The presence of NOM increased the quantity of S-FLG in these cells. Exposure to L-FLG or S-FLG also had a significantly different impact on the intestinal microbial community structure.
Project description:Understanding the colloidal stability of graphene is essential for predicting its transport and ecological risks in aquatic environments. We investigated the agglomeration of 14C-labeled few-layer graphene (FLG) at concentrations spanning nearly four orders of magnitude (2 ?g/L to 10 mg/L) using dynamic light scattering and sedimentation measurements. FLG agglomerates formed rapidly in deionized water at concentrations >3 mg/L. From 1 mg/L to 3 mg/L, salt-induced agglomeration was decreased with dilution of FLG suspensions; the critical coagulation concentration of the more concentrated suspension (3 mg/L) was significantly lower than the dilute suspension (1 mg/L) in the presence of NaCl (1.6 mmol/L and 10 mmol/L, respectively). In contrast, FLG underwent slow agglomeration and settling at concentrations ?0.1 mg/L in NaCl solutions and ambient waters with low ionic strength (<10 mmol/L). FLG nanoparticles with smaller lateral sizes (25 nm-75 nm) were shown to agglomerate more slowly than larger FLG, and these small FLG particles exhibited greater bioaccumulation in zebrafish embryo and stronger chorion penetration ability than larger FLG particles. These findings suggest that FLG at more environmentally relevant concentration is relatively stable and may have implications for exposure of small FLG to ecological receptors.
Project description:While graphene has substantial commercial promise, numerous aspects regarding its ecological effects such as its potential for bioaccumulation are not well known. 14C-labeled few layer graphene (FLG) was dispersed in artificial freshwater and uptake of FLG by Limnodrilus hoffmeisteri, an oligochaete, was assessed. After exposure for 36 h to a 1 mg/L FLG suspension, the FLG body burden in the organism was nearly 60 ng/mg (on a dry mass basis). Multiple characterization results confirmed that the proteins secreted by the organisms during the exposure period coated the FLG, thus increasing its stability and decreasing its size in suspension. Uptake behaviors of Eisenia foetida exposed to FLG and protein-coated FLG at concentrations of approximately 1 mg/kg or to Daphnia magna at 100 ?g/L were also quantified. Protein-coated FLG demonstrated different bioaccumulation behaviors for both organisms compared to uncoated FLG, with the FLG body burden in E. foetida increased but that in D. magna reduced. The data provide the first evidence that the proteins secreted by Limnodrilus hoffmeisteri after exposure to FLG can coat FLG, thus increasing the aqueous stability of FLG, decreasing its size, and changing its bioaccumulation potential.
Project description:The potential human health risks from graphene inhalation exposure have attracted substantial scientific interest as a result of the numerous exciting potential commercial applications of graphene. However, the long-term distribution of graphene in organisms after inhalation is unknown, largely as a result of challenges associated with accurate graphene quantification.Carbon-14 labeled FLG was used to quantify the in vivo distribution of FLG in mice after oral gavage or intratracheal instillation for up to 3 or 28 days after exposure, respectively.Intratracheally instilled FLG was mainly retained in the lung with 47% remaining after 4 weeks. Exposure to non-labeled FLG resulted in dose-dependent acute lung injury and pulmonary edema, but these effects were alleviated with time despite the continued presence of FLG in the lungs. One percent and 0.18% of the intratracheally instilled FLG was present in the liver and spleen, respectively, after 14 days by passing through the air-blood barrier, a finding supported by the results of oral gavage experiments which did not show detectable absorption through the gastrointestinal tract. In addition, 46.2% of the intratracheally instilled FLG was excreted through the feces 28 d after exposure.Quantitative measurements revealed the elimination mechanism for FLG and its biodistribution for two exposure pathways. Graphene persistence in the lung only caused transient pulmonary effects. The in vivo distribution, elimination, and toxicity results provided here measured using a robust quantitative method support the human health risk assessment of graphene.
Project description:Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24?h, after 72?h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC<sub>50</sub>s of 62.8??g/mL (WST-8 assay) and 45.5??g/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC<sub>50</sub>s of 5.4 and 2.9??g/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.
Project description:We investigate the optoelectronic properties of novel graphene/FeCl3-intercalated few-layer graphene (FeCl3-FLG, dubbed graphexeter) heterostructures using photovoltage spectroscopy. We observe a prominent photovoltage signal generated at the graphene/FeCl3-FLG and graphene/Au interfaces, whereas the photovoltage at the FeCl3-FLG/Au interface is negligible. The sign of the photovoltage changes upon sweeping the chemical potential of the pristine graphene through the charge neutrality point, and we show that this is due to the photothermoelectric effect. Our results are a first step toward all-graphene-based photodetectors and photovoltaics.
Project description:In the frame of graphene-based material (GBM) hazard characterization, particular attention should be given to the cutaneous effects. Hence, this study investigates if HaCaT skin keratinocytes exposed to high concentrations of few-layer graphene (FLG) or partially dehydrated graphene oxide (d-GO) for a short time can recover from the cytotoxic insult, measured by means of cell viability, mitochondrial damage and oxidative stress, after GBM removal from the cell medium. When compared to 24 or 72 h continuous exposure, recovery experiments suggest that the cytotoxicity induced by 24 h exposure to GBM is only partially recovered after 48 h culture in GBM-free medium. This partial recovery, higher for FLG as compared to GO, is not mediated by autophagy and could be the consequence of GBM internalization into cells. The ability of GBMs to be internalized inside keratinocytes together with the partial reversibility of the cellular damage is important in assessing the risk associated with skin exposure to GBM-containing devices.
Project description:This study presents a series of short-term studies (total duration 48 h) of uptake and depuration of engineered nanoparticles (ENP) in neonate Daphnia magna. Gold nanoparticles (Au NP) were used to study the influence of size, stabilizing agent and feeding on uptake and depuration kinetics and animal body burdens. 10 and 30 nm Au NP with different stabilizing agents [citrate (CIT) and mercaptoundecanoic acid (MUDA)] were tested in concentrations around 0.5 mg Au/L. Fast initial uptake was observed for all studied Au NP, with CIT stabilized Au NP showing similar rates independent of size and MUDA showing increased uptake for the smaller Au NP (MUDA 10 nm > CIT 10 nm, 30 nm > MUDA 30 nm). However, upon transfer to clean media no clear trend on depuration rates was found in terms of stabilizing agent or size. Independent of stabilizing agent, 10 nm Au NP resulted in higher residual whole-animal body burdens after 24 h depuration than 30 nm Au NP with residual body burdens about one order of magnitude higher of animals exposed to 10 nm Au NP. The presence of food (P. subcapitata) did not significantly affect the body burden after 24 h of exposure, but depuration was increased. While food addition is not necessary to ensure D. magna survival in the presented short-term test design, the influence of food on uptake and depuration kinetics is essential to consider in long term studies of ENP where food addition is necessary. This study demonstrates the feasibility of a short-term test design to assess the uptake and depuration of ENP in D. magna. The findings underlines that the assumptions behind the traditional way of quantifying bioconcentration are not fulfilled when ENPs are studied.
Project description:Graphene family materials (GFMs) are extensively explored for various biomedical applications due to their unique physical properties. The prime challenge is to establish a conclusive safety profile of these nanomaterials and their respective products or devices. Formulating GFMs with appropriate ingredients (e.g., surfactant/compatibilizer) will help to disperse them homogeneously (i.e., within the polymer matrix in the case of polymer-graphene nanocomposites) and aid in good interfacial interaction to achieve the desired properties. However, no cytotoxicity report is available on the effects of the additives on graphene and its incorporated materials. Here, we report in vitro cytotoxicity of formulated FLG (FLG-C), i.e., a mixture of FLG, melamine, and sodium poly(naphthalene sulfonate) (SPS), along with natural rubber (NR) latex and FLG-C-included NR latex nanocomposite (FLG-C-NR) thin films on human vaginal epithelial (HVE) cells. FLG-C shows reduced cellular proliferation (?55%) only at a longer exposure time (72 h) even at a low concentration (50 ?g/mL). It also displays significant down- and upregulation in mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively, whereas no changes are observed in lactate dehydrogenase (LDH), propidium iodide (PI), uptake, and cell cycle analysis at 48 h. In vitro experiments on NR latex and FLG-C-NR latex thin films demonstrate that the incorporation of FLG-C does not compromise the biocompatibility of the NR latex. Further substantiation from the in vivo experiments on the thin films recommends that FLG-C could be suitable to prepare a range of biocompatible rubber latex nanocomposites-based products, viz., next-generation condoms (male and female), surgical gloves, catheters, vaginal rings, bladder-rectum spacer balloon, etc.
Project description:: Graphene-related materials (GRMs) are widely used in various applications due to their unique properties. A growing number of reports describe the impact of different carbon nanomaterials, including graphene oxide (GO), reduced GO (rGO), and carbon nanotubes (CNT), on immune cells, but there is still a very limited number of studies on graphene. In this work, we investigated the biological responses of few layer graphene (FLG) on mouse macrophages (bone marrow derived macrophages, BMDMs), which are part of the first line of defense in innate immunity. In particular, our paper describes our findings of short-term FLG treatment in BMDMs with a focus on observing material internalization and changes in general cell morphology. Subsequent investigation of cytotoxicity parameters showed that increasing doses of FLG did not hamper the viability of cells and did not trigger inflammatory responses. Basal level induced autophagic activity sufficed to maintain the cellular homeostasis of FLG treated cells. Our results shed light on the impact of FLG on primary macrophages and show that FLG does not elicit immunological responses leading to cell death.
Project description:We determined the respiratory uptake and depuration kinetics of perfluorooctanesulfonate (PFOS) in Perinereis wilsoni, a polychaete sandworm used as a model species to investigate the fate of chemical pollutants in coastal environments. The sandworms were kept in gravel-packed containers, and the water levels were varied cyclically to mimic the tides. We used seawater kept at 17.1°C. A 7-day exposure period was followed by a 9-day depuration period. The dissolved PFOS concentration averaged 28 ng/L during the exposure period. Sandworm samples were collected regularly for analysis of PFOS concentrations, and a first-order-kinetics model was applied to the concentrations. The respiratory absorption efficiency of PFOS was estimated to be 11% that of oxygen, which is higher than the corresponding estimates reported for several fish species. The estimated depuration half-life of 15 days was comparable to previously reported estimates for fish and oligochaete species. The bioconcentration factor was 470.