Chronic Engineered Nanoparticle Additions Alter Insect Emergence and Result in Metal Flux from Aquatic Ecosystems into Riparian Food Webs.

Freshwater ecosystems are exposed to engineered nanoparticles (NPs) through discharge from wastewater and agricultural runoff. We conducted a 9-month mesocosm experiment to examine the combined effects of chronic NP additions on insect emergence and insect-mediated contaminant flux to riparian spiders. Two NPs (copper, gold, plus controls) were crossed by two levels of nutrients in 18 outdoor mesocosms open to natural insect and spider colonization. We collected adult insects and two riparian spider genera, Tetragnatha and Dolomedes, for 1 week on a monthly basis. We estimated a significant decrease in cumulative insect emergence of 19% and 24% after exposure to copper and gold NPs, irrespective of nutrient level. NP treatments led to elevated copper and gold tissue concentrations in adult insects, which resulted in terrestrial fluxes of metals. These metal fluxes were associated with increased gold and copper tissue concentrations for both spider genera. We also observed about 25% fewer spiders in the NP mesocosms, likely due to reduced insect emergence and/or NP toxicity. These results demonstrate the transfer of NPs from aquatic to terrestrial ecosystems via emergence of aquatic insects and predation by riparian spiders, as well as significant reductions in insect and spider abundance in response to NP additions.

Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells.

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl2 (ions) at doses between 0.1 and 30 ug/ml (approximately the no observable adverse effect level to a high degree of cytotoxicity). Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. With nano Cu and nano CuO, few indications of cytotoxicity were observed between 0.1 and 3 ug/ml. In respect to dose, lactate dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity parameters. The next most responsive parameters were alanine aminotransferase, glutathione reductase, glucose 6-phosphate dehydrogenase, and protein concentration. The medium responsive parameters were superoxide dismutase, gamma glutamyltranspeptidase, total bilirubin, and microalbumin. The parameters glutathione peroxidase, glutathione reductase, and protein were all altered by nano Cu and nano CuO but not by CuCl2 exposures. Our chief observations were (1) significant decreases in glucose 6-phosphate dehydrogenase and glutathione reductase was observed at doses below the doses that show high cytotoxicity, (2) even high cytotoxicity did not induce large changes in some study parameters (e.g., alkaline phosphatase, catalase, microalbumin, total bilirubin, thioredoxin reductase, and triglycerides), (3) even though many significant biochemical effects happen only at doses showing varying degrees of cytotoxicity, it was not clear that cytotoxicity alone caused all of the observed significant biochemical effects, and (4) the decreased glucose 6-phosphate dehydrogenase and glutathione reductase support the view that oxidative stress is a main toxicity pathway of CuCl2 and Cu-containing nanomaterials.

Copper and Gold Nanoparticles Increase Nutrient Excretion Rates of Primary Consumers.

Freshwater ecosystems are exposed to engineered nanoparticles through municipal and industrial wastewater-effluent discharges and agricultural nonpoint source runoff. Because previous work has shown that engineered nanoparticles from these sources can accumulate in freshwater algal assemblages, we hypothesized that nanoparticles may affect the biology of primary consumers by altering the processing of two critical nutrients associated with growth and survivorship, nitrogen and phosphorus. We tested this hypothesis by measuring the excretion rates of nitrogen and phosphorus of Physella acuta, a ubiquitous pulmonate snail that grazes heavily on periphyton, exposed to either copper or gold engineered nanoparticles for 6 months in an outdoor wetland mesocosm experiment. Chronic nanoparticle exposure doubled nutrient excretion when compared to the control. Gold nanoparticles increased nitrogen and phosphorus excretion rates more than copper nanoparticles, but overall, both nanoparticles led to higher consumer excretion, despite contrasting particle stability and physiochemical properties. Snails in mesocosms enriched with nitrogen and phosphorus had overall higher excretion rates than ones in ambient (no nutrients added) mesocosms. Stimulation patterns were different between nitrogen and phosphorus excretion, which could have implications for the resulting nutrient ratio in the water column. These results suggest that low concentrations of engineered nanoparticles could alter the metabolism of consumers and increase consumer-mediated nutrient recycling rates, potentially intensifying eutrophication in aquatic systems, for example, the increased persistence of algal blooms as observed in our mesocosm experiment.

Biochemical Effects of Silver Nanomaterials in Human Hepatocellular Carcinoma (HepG2) Cells.

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed to between 0.01 and 300 ug/ml of different silver nanomaterials and AgNO3 for 3 days. Treatment chemicals included a custom synthesized rod shaped nano Ag, a glutathione capped nano Ag, polyvinylpyrrolidone (PVP) capped nano Ag (75 nm) from Nanocomposix and AgNO3. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function and oxidative stress. Few indications of cytotoxicity were observed between 0.1 ug/ml and 6 ug/ml of any nano Ag. At 10 ug/ml and above, Ag containing nanomaterials caused a moderate to severe degree of cytotoxicity in HepG2 cells. Lactate dehydrogenase and aspartate transaminase activity alterations were the most sensitive cytotoxicity parameters. Some biochemical parameters were altered by exposures to both nano Ag and AgNO3 (statistically significant increases in alkaline phosphatase, gamma glutamyltranspeptidase, glutathione peroxidase and triglycerides; in contrast both glutathione reductase and HepG2 protein concentration were both decreased). Three parameters were significantly altered by nano Ag but not by AgNO3 (decreases in glucose 6-phosphate dehydrogenase and thioredoxin reductase and increases in catalase). Cytotoxicity per se did not appear to fully explain the patterns of biological responses observed. Some of the observations with the three nano Ag (increases in alkaline phosphatase, catalase, gamma glutamyltranspeptidase, as well as decreases in glucose 6-phosphate dehydrogenase and glutathione reductase) are in the same direction as HepG2 responses to other nanomaterials composed of TiO2, CeO2, SiO2, CuO and Cu. Therefore, these biochemical responses may be due to micropinocytosis of nanomaterials, membrane damage, oxidative stress and/or cytotoxicity. Decreased G6PDH (by all three nano Ag forms) and GRD activity (only nano Ag R did not cause decreases) support and are consistent with the oxidative stress theory of Ag nanomaterial action.

Differential Reactivity of Copper- and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm.

Reliable predictions of the environmental fate and risk of engineered nanomaterials (ENMs) require a better understanding of ENM reactivity in complex, biologically active systems for chronic low-concentration exposure scenarios. Here, simulated freshwater wetland mesocosms were dosed with ENMs to assess how their reactivity and seasonal changes in environmental parameters influence ENM fate in aquatic systems. Copper-based ENMs (Kocide), known to dissolve in water, and gold nanoparticles (AuNPs), stable against dissolution in the absence of specific ligands, were added weekly to mesocosm waters for 9 months. Metal accumulation and speciation changes in the different environmental compartments were assessed over time. Copper from Kocide rapidly dissolved likely associating with organic matter in the water column, transported to terrestrial soils and deeper sediment where it became associated with organic or sulfide phases. In contrast, Au accumulated on/in the macrophytes where it oxidized and transferred over time to surficial sediment. A dynamic seasonal accumulation and metal redox cycling were found between the macrophyte and the surficial sediment for AuNPs. These results demonstrate the need for experimental quantification of how the biological and chemical complexity of the environment, combined with their seasonal variations, drive the fate of metastable ENMs.

Biochemical effects of some CeO2, SiO2, and TiO2 nanomaterials in HepG2 cells.

The potential mammalian hepatotoxicity of nanomaterials was explored in dose-response and structure-activity studies in human hepatic HepG2 cells exposed to between 10 and 1000 µg/ml of five different CeO2, three SiO2, and one TiO2-based particles for 3 days. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. Few indications of cytotoxicity were observed between 10 and 30 µg/ml. In the 100 to 300 µg/ml exposure range, a moderate degree of cytotoxicity was often observed. At 1000 µg/ml exposures, all but TiO2 showed a high degree of cytotoxicity. Cytotoxicity per se did not seem to fully explain the observed patterns of biochemical parameters. Four nanomaterials (all three SiO2) decreased glucose 6-phosphate dehydrogenase activity with some significant decreases observed at 30 µg/ml. In the range of 100 to 1000 µg/ml, the activities of glutathione reductase (by all three SiO2) and glutathione peroxidase were decreased by some nanomaterials. Decreased glutathione concentration was also found after exposure to four nanomaterials (all three nano SiO2 particles). In this study, the more responsive and informative assays were glucose 6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, lactate dehydrogenase, and aspartate transaminase. In this study, there were six factors that contribute to oxidative stress observed in nanomaterials exposed to hepatocytes (decreased glutathione content, reduced glucose 6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and increased catalase activities). With respect to structure-activity, nanomaterials of SiO2 were more effective than CeO2 in reducing glutathione content, glucose 6-phosphate dehydrogenase, glutathione reductase, and superoxide dismutase activities.

Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms.

Trace metals associated with nanoparticles are known to possess reactivities that are different from their larger-size counterparts. However, the relative importance of small relative to large particles for the overall distribution and biouptake of these metals is not as well studied in complex environmental systems. Here, we have examined differences in the long term fate and transport of ceria (CeO2) nanoparticles of two different sizes (3.8 vs 185 nm), dosed weekly to freshwater wetland mesocosms over 9 months. While the majority of CeO2 particles were detected in soils and sediments at the end of nine months, there were significant differences observed in fate, distribution, and transport mechanisms between the two materials. Small nanoparticles were removed from the water column primarily through heteroaggregation with suspended solids and plants, while large nanoparticles were removed primarily by sedimentation. A greater fraction of small particles remained in the upper floc layers of sediment relative to the large particles (31% vs 7%). Cerium from the small particles were also significantly more bioavailable to aquatic plants (2% vs 0.5%), snails (44 vs 2.6 ng), and insects (8 vs 0.07 µg). Small CeO2 particles were also significantly reduced from Ce(IV) to Ce(III), while aquatic sediments were a sink for untransformed large nanoparticles. These results demonstrate that trace metals originating from nanoscale materials have much greater potential than their larger counterparts to distribute throughout multiple compartments of a complex aquatic ecosystem and contribute to the overall bioavailable pool of the metal for biouptake and trophic transfer.

Validation of a Sulfuric Acid Digestion Method for Inductively Coupled Plasma Mass Spectrometry Quantification of TiO2 Nanoparticles.

A consistent analytical method incorporating sulfuric acid (H2SO4) digestion and ICP-MS quantification has been developed for TiO2 quantification in biotic and abiotic environmentally relevant matrices. Sample digestion in H2SO4 at 110°C provided consistent results without using hydrofluoric acid or microwave digestion. Analysis of seven replicate samples for four matrices on each of 3 days produced Ti recoveries of 97% ± 2.5%, 91 % ± 4.0%, 94% ± 1.8%, and 73 % ± 2.6% (mean ± standard deviation) from water, fish tissue, periphyton, and sediment, respectively. The method demonstrated consistent performance in analysis of water collected over a 1 month.

Titanium dioxide nanoparticle exposure reduces algal biomass and alters algal assemblage composition in wastewater effluent-dominated stream mesocosms.

A 5-week mesocosm experiment was conducted to investigate the toxicity of titanium dioxide nanoparticles (TiO2NPs) to periphytic algae in an environmentally-realistic scenario. We used outdoor experimental streams to simulate the characteristics of central Texas streams receiving large discharges of wastewater treatment plant effluent during prolonged periods of drought. The streams were continually dosed and maintained at two concentrations. The first represents an environmentally relevant concentration of 0.05 mg L-1 (low concentration). The second treatment of 5 mg L-1 (high concentration) was selected to represent a scenario where TiO2NPs are used for photocatalytic degradation of pharmaceuticals in wastewater. Algal cell density, chlorophyll-a, ash-free dry mass, algal assemblage composition, and Ti accumulation were determined for the periphyton in the riffle sections of each stream. The high concentration treatment of TiO2NPs significantly decreased algal cell density, ash-free dry mass, and chlorophyll-a, and altered algal assemblage composition. Decreased abundance of three typically pollution-sensitive taxa and increased abundance of two genera associated with heavy metal sorption and organic pollution significantly contributed to algal assemblage composition changes in response to TiO2NPs. Benefits of the use of TiO2NPs in wastewater treatment plants will need to be carefully weighed against the demonstrated ability of these NPs to cause large changes in periphyton that would likely propagate significant effects throughout the stream ecosystem, even in the absence of direct toxicity to higher trophic level organisms.

Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells.

BACKGROUND: To better assess potential hepatotoxicity of nanomaterials, human liver HepG2 cells were exposed for 3 days to five different CeO2 (either 30 or 100 µg/ml), 3 SiO2 based (30 µg/ml) or 1 CuO (3 µg/ml) nanomaterials with dry primary particle sizes ranging from 15 to 213 nm. Metabolomic assessment of exposed cells was then performed using four mass spectroscopy dependent platforms (LC and GC), finding 344 biochemicals. RESULTS: Four CeO2, 1 SiO2 and 1 CuO nanomaterials increased hepatocyte concentrations of many lipids, particularly free fatty acids and monoacylglycerols but only CuO elevated lysolipids and sphingolipids. In respect to structure-activity, we now know that five out of six tested CeO2, and both SiO2 and CuO, but zero out of four TiO2 nanomaterials have caused this elevated lipids effect in HepG2 cells. Observed decreases in UDP-glucuronate (by CeO2) and S-adenosylmethionine (by CeO2 and CuO) and increased S-adenosylhomocysteine (by CuO and some CeO2) suggest that a nanomaterial exposure increases transmethylation reactions and depletes hepatic methylation and glucuronidation capacity. Our metabolomics data suggests increased free radical attack on nucleotides. There was a clear pattern of nanomaterial-induced decreased nucleotide concentrations coupled with increased concentrations of nucleic acid degradation products. Purine and pyrimidine alterations included concentration increases for hypoxanthine, xanthine, allantoin, urate, inosine, adenosine 3',5'-diphosphate, cytidine and thymidine while decreases were seen for uridine 5'-diphosphate, UDP-glucuronate, uridine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-monophophate, cytidine 5'-monophosphate and cytidine 3'-monophosphate. Observed depletions of both 6-phosphogluconate, NADPH and NADH (all by CeO2) suggest that the HepG2 cells may be deficient in reducing equivalents and thus in a state of oxidative stress. CONCLUSIONS: Metal oxide nanomaterial exposure may compromise the methylation, glucuronidation and reduced glutathione conjugation systems; thus Phase II conjugational capacity of hepatocytes may be decreased. This metabolomics study of the effects of nine different nanomaterials has not only confirmed some observations of the prior 2014 study (lipid elevations caused by one CeO2 nanomaterial) but also found some entirely new effects (both SiO2 and CuO nanomaterials also increased the concentrations of several lipid classes, nanomaterial induced decreases in S-adenosylmethionine, UDP-glucuronate, dipeptides, 6-phosphogluconate, NADPH and NADH).

Relative Contributions of Copper Oxide Nanoparticles and Dissolved Copper to Cu Uptake Kinetics of Gulf Killifish (Fundulus grandis) Embryos.

The toxicity of soluble metal-based nanomaterials may be due to the uptake of metals in both dissolved and nanoparticulate forms, but the relative contributions of these different forms to overall metal uptake rates under environmental conditions are not quantitatively defined. Here, we investigated the linkage between the dissolution rates of copper(II) oxide (CuO) nanoparticles (NPs) and their bioavailability to Gulf killifish (Fundulus grandis) embryos, with the aim of quantitatively delineating the relative contributions of nanoparticulate and dissolved species for Cu uptake. Gulf killifish embryos were exposed to dissolved Cu and CuO NP mixtures comprising a range of pH values (6.3-7.5) and three types of natural organic matter (NOM) isolates at various concentrations (0.1-10 mg-C L-1), resulting in a wide range of CuO NP dissolution rates that subsequently influenced Cu uptake. First-order dissolution rate constants of CuO NPs increased with increasing NOM concentration and for NOM isolates with higher aromaticity, as indicated by specific ultraviolet absorbance (SUVA), while Cu uptake rate constants of both dissolved Cu and CuO NP decreased with NOM concentration and aromaticity. As a result, the relative contribution of dissolved Cu and nanoparticulate CuO species for the overall Cu uptake rate was insensitive to NOM type or concentration but largely determined by the percentage of CuO that dissolved. These findings highlight SUVA and aromaticity as key NOM properties affecting the dissolution kinetics and bioavailability of soluble metal-based nanomaterials in organic-rich waters. These properties could be used in the incorporation of dissolution kinetics into predictive models for environmental risks of nanomaterials.

Differential Genomic Effects of Six Different TiO2 Nanomaterials on Human Liver HepG2 Cells.

Human HepG2 cells were exposed to six TiO2 nanomaterials (with dry primary particle sizes ranging from 22 to 214 nm, either 0.3, 3, or 30 µg/mL) for 3 days. Some of these canonical pathways changed by nano-TiO2 in vitro treatments have been already reported in the literature, such as NRF2-mediated stress response, fatty acid metabolism, cell cycle and apoptosis, immune response, cholesterol biosynthesis, and glycolysis. But this genomic study also revealed some novel effects such as protein synthesis, protein ubiquitination, hepatic fibrosis, and cancer-related signaling pathways. More importantly, this genomic analysis of nano-TiO2 treated HepG2 cells linked some of the in vitro canonical pathways to in vivo adverse outcomes: NRF2-mediated response pathways to oxidative stress, acute phase response to inflammation, cholesterol biosynthesis to steroid hormones alteration, fatty acid metabolism changes to lipid homeostasis alteration, G2/M cell checkpoint regulation to apoptosis, and hepatic fibrosis/stellate cell activation to liver fibrosis.

Press or pulse exposures determine the environmental fate of cerium nanoparticles in stream mesocosms.

Risk-assessment models indicate that stream ecosystems receiving municipal wastewater effluent may have the greatest potential for exposure to manufactured nanoparticles. The authors determined the fate of cerium oxide (CeO2 ) nanoparticles in outdoor stream mesocosms using 1) 1-time pulse addition of CeO2 nanoparticles, representative of accidental release, and 2) continuous, low-level press addition of CeO2 nanoparticles, representative of exposure via wastewater effluent. The pulse addition led to rapid nanoparticle floc formation, which appeared to preferentially deposit on periphyton in low-energy areas downstream from the location of the input, likely as a result of gravitational sedimentation. Floc formation limited the concentration of suspended nanoparticles in stream water to <5% of target and subsequent downstream movement. In contrast, press addition of nanoparticles led to higher suspended nanoparticle concentrations (77% of target) in stream water, possibly as a result of stabilization of suspended nanoparticles through interaction with dissolved organic carbon. Smaller nanoparticle aggregates appeared to preferentially adsorb to stream surfaces in turbulent sections, where Ce concentrations were highest in the press, likely a result of stochastic encounter with the surface. Streams receiving wastewater effluent containing nanoparticles may lead to exposure of aquatic organisms over a greater spatial extent than a similar amount of nanoparticles from an accidental release. Exposure models must take into account these mechanisms controlling transport and depositional processes.

Differential Genomic Effects on Signaling Pathways by Two Different CeO2 Nanoparticles in HepG2 Cells.

To investigate genomic effects, human liver hepatocellular carcinoma (HepG2) cells were exposed for three days to two different forms of nanoparticles both composed of CeO2 (0.3, 3 and 30 µg/mL). The two CeO2 nanoparticles had dry primary particle sizes of 8 nanometers {(M) made by NanoAmor} and 58 nanometers {(L) made by Alfa Aesar} and differ in various other physical-chemical properties as well. The smaller particle has stronger antioxidant properties, probably because it has higher Ce3+ levels on the particle surface, as well as more surface area per unit weight. Nanoparticle M showed a normal dose-response pattern with 363, 633 and 1273 differentially expressed genes (DEGs) at 0.3, 3 and 30 µg/mL, respectively. In contrast, nanoparticle L showed a puzzling dose-response pattern with the most DEGs found in the lowest exposure group with 1049, 303 and 323 DEGs at 0.3, 3 and 30 µg/mL, respectively. This systems biological genomic study showed that the major altered pathways by these two nano cerium oxides were protein synthesis, stress response, proliferation/cell cycle, cytoskeleton remodeling/actin polymerization and cellular metabolism. Some of the canonical pathways affected were mTOR signaling, EIF2 signaling, fatty acid activation, G2/M DNA damage checkpoint regulation, glycolysis and protein ubiquitination. These two CeO2 nanoparticles differed considerably in their genomic effects. M is more active than L in respect to altering the pathways of mitochondrial dysfunction, acute phase response, apoptosis, 14-3-3 mediated signaling, remodeling of epithelial adherens junction signaling, actin nucleation by ARP-WASP complex, altered TCA cycle and elevated fatty acid concentrations by metabolomics. However, L is more active than M in respect to the pathways of NRF2-mediated stress response and hepatic fibrosis/hepatic stellate cell activation. One major difference in the cell response to nano M and L is that nano M caused the Warburg effect while nano L did not.