Theme 3: Ecosystem Functional Responses to Nanomaterials

Macroscopic and particle scale understanding of the effects of adsorbed organic macromolecules on NP-bacteria interactions and toxicity


We have previously demonstrated (2010) that coatings on ENP are a key determinate of the interactions between bacterial cells and ENP as well as the expression of toxicity by the ENP.  We showed that the toxicity of nano Fe0 (NZVI), which is only observed during cell-ENP contact, was prevented by both engineered and natural organic matter (NOM) coatings on NZVI.  TEM microscopy revealed that the coatings prevented direct contact between NZVI and the cells.  The implication is that the coatings prevent ENP-cell contact through electrosteric and/or electrostatic mechanisms. 

While both natural and engineered coatings decreased the toxicity of NZVI towards cells, in the case of TiO2 ENP, polymer coatings had little effect on the toxic behavior while NOM disrupted toxicity (Li et al, in review).  The mechanism of this coating-specific disruption of toxicity was linked to scavenging of reactive oxygen species by NOM and is likely the result of aromatic groups in NOM that are absent in the engineered polymer coatings.  These findings together highlighted the importance of understanding the mechanisms of toxicity as well as the mechanisms by which coatings interfere with the toxic expression of ENP.

We recently addressed the impact of coatings on the toxicity of AgNP, an ENP that, unlike NZVI which is toxic through direct attachment to cells and TiO2 which produce (photochemically) toxic, reactive oxygen species, produce a toxic silver ion through dissolution in aqueous media. Surprisingly, disruption of AgNP cytotoxicity was only observed when NOM was added in the aqueous phase, independent of AgNP addition and not when present as a coating on AgNP.  AgNP were toxic towards E. coli with polymer regardless of whether it was added as a coating or in the aqueous phase.  Studies with silver ion alone show that NOM in solution reduced its cytotoxicity by reducing the concentration of Ag+.  Addition of NOM with higher sulfur content increased the protective effect of NOM.  This finding suggests that NOM is protective of cells from AgNP toxicity by scavenging toxic silver ion on sulfur groups associated with the NOM.  Taken together, our work with the coatings show that the type of macromolecule, and not merely its presence) is important for determining the toxicity of ENP.  Moreover, the results show that understanding the mechanisms of toxicity expression and the disruptive mechanisms of the coatings will be important for determining the risks associated with ENP in the environment as well as the engineering of relatively benign particles. 

Ecosystem impacts of nanomaterials


CEINT 2010-2011 Wetland Mesocosm Experiment:

Experimental overview:

Our largest endeavour has been the ongoing monitoring of treatment effects in a replicated experiment using 24 slantboard wetland mesocosms. These mesocosms were constructed to allow us to examine the impact of silver nanoparticles in wetland ecosystems in a fully replicated experiment. Planted in March of 2010, these 4’x12’ mesocosms consist of an upland, aquatic, and periodically flooded area. The slantboard design allows examination of the fate, transport, and impacts of nanomaterials in a wide range of conditions in a single experimental unit. The 24 mesocosms were planted with 6 wetland plant species, and 4 macrophytes. The open design also allowed recruitment of many different taxa of intvertebrates and algae.

The mesocosms were dosed on August 17th, 2010, and the experiment is ongoing. There are six treatments: three silver with final concentrations in the water of 2.5mg Ag/L (gum arabic coated AgNPs, GA-AgNPs; polyvinylpyrrolidone coated AgNPs, PVP-AgNPs; and AgNO3), and three control with no added silver (control, GA coating control, and PVP coating control). To account for the NO3- added in the AgNO3 treatment, an equivalent amount of KNO3 was added to all mesocosms in the replicated experiment. There were 3 replicates of all treatments with the exception of the control treatment, which had 4 replicates. Of the 24 mesocosms established, 19 were included in the fully replicated silver experiment. The other five were used in an unreplicated study of nanomaterial fate and transport by the Wiesner lab.

Early impacts:

After dosing with silver, the aquatic macrophytes senesced in silver treated mesocosms. Likely from the senescing macrophytes, there was a pulse of dissolved organic C (DOC), and small increases in the dissolved nutrients NH4+, NO3-, and phosphate. These impacts were strongest in the GA-AgNP and AgNO3 treatments, with less pronounced increases in the PVP-AgNP treatment. Associated with this increase in DOC and loss of macrophyte biomass, there was also a large decrease in dissolved O2 in the GA-AgNP and AgNO3 treatments from increased respiration and decreased photosynthesis.

The most intriguing ecosystem functional response was a 2-3 order of magnitude increase in methane concentration in the watercolumn in silver amended treatments (day 0 being the start of the experiment). This was not simply a result from increased DOC and decreased dissolved O2; both GA-AgNPs and AgNO3 had stronger impacts on DOC and O2 than PVP-AgNPs, yet all three treatments had similar impacts on methane production. Similar results have been observed by our group in laboratory experiments, and further work is planned to see what exactly causes this increase in methane concentration.

Isolation characterization techniques for NPs in environmental media


We have developed and begun to optimize techniques that utilize asymmetrical flow field flow fractionation with multi-angle laser light scattering, UV-Vis spectroscopy, and inductively coupled plasma mass spectrometry detection for in situ characterization of Ag NP aggregation state in aqueous samples including soil and sediment pore water. Initial results demonstrate the ability of the technique to characterize Ag NPs in soil pore water and demonstrate differences in partitioning-aggregation  between citrate and PVP capped Ag-NPs.

The bioavailability of nanoparticles to plants and the potential for trophic transfer in simple terrestrial food chains


Plant uptake studies have revealed that there are no significant differences in uptake of Au-NPs based on size or relative hydrophobicity of the surface coating, although spatial analysis by laser ablation ICP-MS of leaf tissues reveals heterogeneous distribution of the larger particles (30-50 nm) in the leaves, being concentrated in larger vasulature. This may have implications to the trophic transfer of plant accumulated NPs to herbivorous consumers. No uptake in wheat was observed for any treatment, suggesting important species differences in the bioavailability of NPs.

Initial studies on model ion-loaded membrane vesicles demonstrate that both tannic acid and citrate capped Au-NPs induce significant ion (proton) leakage due to the disruption of phospholipid arrangement for vesicles of two different compositions (plant derived-azolectin and plant and fungal mixture azolectin and ergosterol). However, the vesicles with ergosterol were far more sensitive to all particles and displayed surface functionalization dependent differences in proton leakage, with much greater leakage observed for the citrate capped Au-NPs.

Properties and Impacts of Nanoparticles on Microbial Communities


Impacts ofAgNPs on ammonia oxidizing bacteria (AOB). The impacts of 0.2, 2 and 20 ppm AgNPs coated with citrate, gum arabic, and polyvinylpyrrolidone (PVP) were investigated on the model AOB Nitrosomonas europaea. Specifically, the inhibition of nitrite production was quantified and follow up studies were undertaken to determine the mechanisms of inhibition for each AgNP. Nitrite production was lowered by 68 ± 4 and 96 ± 1 % for citrate and gum arabic coated AgNPs, respectively, at a concentration of 2 ppm while no significant effect was observed at 0.2 ppm. At 20 ppm of citrate and gum arabic coated AgNPs, a reduction in nitrite production was measured of 85 ± 1 and 93 ± 1.4 %, respectively. Citrate AgNP treatments were statistically different between 2 and 20 ppm, while gum arabic AgNPs were not. For PVP coated AgNPs, nitrite production was not as greatly affected until reaching 20 ppm, where a 89 ± 1% reduction was measured. Lower concentrations tested of PVP AgNPs did not exhibit statistical differences from no-silver control samples. The membrane disruption of N. europaea exposed to citrate and PVP coated AgNPs was present at concentrations, which exhibited nitrite production inhibition, while gum arabic coated AgNP did not affect membrane integrity even at concentrations where inhibition of nitrite production was observed. The addition of L-cysteine relieved all AgNP toxicity in nitrite inhibition and membrane integrity assays, with the exception of the PVP AgNPs. Gene expression studies on amoA, hao, gshB, and merA showed that the only gene that was impacted by AgNPs was merA, which was up-regulated in the presence of all three AgNPs. These results suggest that the infiltration of some AgNPs could negatively impact nitrification and that coating plays a critical role in determining the ultimate nanoparticle toxicity. 


Ecological impacts of AgNPs on soil microbial communities following biosolid land application. To examine the effects of AgNPs on microbial community structure, we compared the composition of bacterial 16S-rRNA genes present using Terminal Restriction Fragment Length Polymorphisms on 0-1cm soils immediately prior to biosolid amendment, immediately following addition and 50 days later. As expected, prior to amendment the composition was similar in all mesocosms. Immediately following addition, there was a large shift in all biosolid treatments away from the Controls likely due to biosolid associated microbes. In addition to all three biosolid treatments being significantly different from Controls, the three slurry treatments were significantly different from one. The differences among treatments decreased by day 50, with controls remaining distinct from slurry treatments, but slurry treatments converging on one another by the end of the experiment, suggesting that the effects of biosolids and silver on community composition decreased over longer time scales.