Bioaccumulation of Multiwall Carbon Nanotubes in <i>Tetrahymena thermophila</i> by Direct Feeding or Trophic Transfer

Consumer goods contain multiwall carbon nanotubes (MWCNTs) that could be released during product life cycles into the environment, where their effects are uncertain. Here, we assessed MWCNT bioaccumulation in the protozoan <i>Tetrahymena thermophila</i> via trophic transfer from bacterial prey (<i>Pseudomonas aeruginosa</i>) versus direct uptake from growth media. The experiments were conducted using ¹⁴C-labeled MWCNT (¹⁴C-MWCNT) doses at or below 1 mg/L, which proved subtoxic since there were no adverse effects on the growth of the test organisms. A novel contribution of this study was the demonstration of the ability to quantify MWCNT bioaccumulation at low (sub μg/kg) concentrations accomplished by employing accelerator mass spectrometry (AMS). After the treatments with MWCNTs at nominal concentrations of 0.01 mg/L and 1 mg/L, <i>P. aeruginosa</i> adsorbed considerable amounts of MWCNTs: (0.18 ± 0.04) μg/mg and (21.9 ± 4.2) μg/mg bacterial dry mass, respectively. At the administered MWCNT dose of 0.3 mg/L, <i>T. thermophila</i> accumulated up to (0.86 ± 0.3) μg/mg and (3.4 ± 1.1) μg/mg dry mass by trophic transfer and direct uptake, respectively. Although MWCNTs did not biomagnify in the microbial food chain, MWCNTs bioaccumulated in the protozoan populations regardless of the feeding regime, which could make MWCNTs bioavailable for organisms at higher trophic levels

Biological Uptake and Depuration of Radio-labeled Graphene by <i>Daphnia magna</i>

Graphene layers are potential candidates in a large number of applications. However, little is known about their ecotoxicological risks largely as a result of a lack of quantification techniques in complex environmental matrices. In this study, graphene was synthesized by means of graphitization and exfoliation of sandwich-like FePO₄/dodecylamine hybrid nanosheets, and ¹⁴C was incorporated in the synthesis. ¹⁴C-labeled graphene was spiked to artificial freshwater and the uptake and depuration of graphene by <i>Daphnia magna</i> were assessed. After exposure for 24 h to a 250 μg/L solution of graphene, the graphene concentration in the organism was nearly 1% of the organism dry mass. These organisms excreted the graphene to clean artificial freshwater and achieved roughly constant body burdens after 24 h depuration periods regardless of the initial graphene exposure concentration. Addition of algae and humic acid to water during the depuration period resulted in release of a significant fraction (>90%) of the accumulated graphene, but some still remained in the organism. Accumulated graphene in adult <i>Daphnia</i> was likely transferred to the neonates. The uptake and elimination results provided here support the environmental risk assessment of graphene and the graphene quantification method is a powerful tool for additional studies

Detection and Quantification of Graphene-Family Nanomaterials in the Environment

An increase in production of commercial products containing graphene-family nanomaterials (GFNs) has led to concern over their release into the environment. The fate and potential ecotoxicological effects of GFNs in the environment are currently unclear, partially due to the limited analytical methods for GFN measurements. In this review, the unique properties of GFNs that are useful for their detection and quantification are discussed. The capacity of several classes of techniques to identify and/or quantify GFNs in different environmental matrices (water, soil, sediment, and organisms), after environmental transformations, and after release from a polymer matrix of a product is evaluated. Extraction and strategies to combine methods for more accurate discrimination of GFNs from environmental interferences as well as from other carbonaceous nanomaterials are recommended. Overall, a comprehensive review of the techniques available to detect and quantify GFNs are systematically presented to inform the state of the science, guide researchers in their selection of the best technique for the system under investigation, and enable further development of GFN metrology in environmental matrices. Two case studies are described to provide practical examples of choosing which techniques to utilize for detection or quantification of GFNs in specific scenarios. Because the available quantitative techniques are somewhat limited, more research is required to distinguish GFNs from other carbonaceous materials and improve the accuracy and detection limits of GFNs at more environmentally relevant concentrations

Biological Uptake, Distribution, and Depuration of Radio-Labeled Graphene in Adult Zebrafish: Effects of Graphene Size and Natural Organic Matter

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 μg/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

Multiple Method Analysis of TiO₂ Nanoparticle Uptake in Rice (<i>Oryza sativa</i> L.) Plants

Understanding the translocation of nanoparticles (NPs) into plants is challenging because qualitative and quantitative methods are still being developed and the comparability of results among different methods is unclear. In this study, uptake of titanium dioxide NPs and larger bulk particles (BPs) in rice plant (<i>Oryza sativa L</i>.) tissues was evaluated using three orthogonal techniques: electron microscopy, single-particle inductively coupled plasma mass spectroscopy (spICP-MS) with two different plant digestion approaches, and total elemental analysis using ICP optical emission spectroscopy. In agreement with electron microscopy results, total elemental analysis of plants exposed to TiO₂ NPs and BPs at 5 and 50 mg/L concentrations revealed that TiO₂ NPs penetrated into the plant root and resulted in Ti accumulation in above ground tissues at a higher level compared to BPs. spICP-MS analyses revealed that the size distributions of internalized particles differed between the NPs and BPs with the NPs showing a distribution with smaller particles. Acid digestion resulted in higher particle numbers and the detection of a broader range of particle sizes than the enzymatic digestion approach, highlighting the need for development of robust plant digestion procedures for NP analysis. Overall, there was agreement among the three techniques regarding NP and BP penetration into rice plant roots and spICP-MS showed its unique contribution to provide size distribution information

Correction to Copper Oxide Nanoparticle Mediated DNA Damage in Terrestrial Plant Models

Correction to Copper Oxide Nanoparticle Mediated DNA Damage in Terrestrial Plant Model

Quantification of Carbon Nanotubes in Environmental Matrices: Current Capabilities, Case Studies, and Future Prospects

Carbon nanotubes (CNTs) have numerous exciting potential applications and some that have reached commercialization. As such, quantitative measurements of CNTs in key environmental matrices (water, soil, sediment, and biological tissues) are needed to address concerns about their potential environmental and human health risks and to inform application development. However, standard methods for CNT quantification are not yet available. We systematically and critically review each component of the current methods for CNT quantification including CNT extraction approaches, potential biases, limits of detection, and potential for standardization. This review reveals that many of the techniques with the lowest detection limits require uncommon equipment or expertise, and thus, they are not frequently accessible. Additionally, changes to the CNTs (e.g., agglomeration) after environmental release and matrix effects can cause biases for many of the techniques, and biasing factors vary among the techniques. Five case studies are provided to illustrate how to use this information to inform responses to real-world scenarios such as monitoring potential CNT discharge into a river or ecotoxicity testing by a testing laboratory. Overall, substantial progress has been made in improving CNT quantification during the past ten years, but additional work is needed for standardization, development of extraction techniques from complex matrices, and multimethod comparisons of standard samples to reveal the comparability of techniques

Agglomeration of <i>Escherichia coli</i> with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard <i>Caenorhabditis elegans</i> Toxicity Assay

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) toxicity assay containing an <i>Escherichia coli</i> (<i>E. coli</i>) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with <i>E. coli</i> cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on <i>C. elegans</i> growth and reproduction. When the ENPs were tested in two alternate <i>C. elegans</i> assays that did not contain <i>E. coli</i>, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays

Agglomeration of <i>Escherichia coli</i> with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard <i>Caenorhabditis elegans</i> Toxicity Assay

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) toxicity assay containing an <i>Escherichia coli</i> (<i>E. coli</i>) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with <i>E. coli</i> cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on <i>C. elegans</i> growth and reproduction. When the ENPs were tested in two alternate <i>C. elegans</i> assays that did not contain <i>E. coli</i>, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays

Separation, Sizing, and Quantitation of Engineered Nanoparticles in an Organism Model Using Inductively Coupled Plasma Mass Spectrometry and Image Analysis

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)­ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake <i>via</i> high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and <i>in vivo</i> quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment