Olga V. Tsyusko

Ecotoxicity test methods for engineered nanomaterials: Practical experiences and recommendations from the bench

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers.

Effect of silver nanoparticle surface coating on bioaccumulation and reproductive toxicity in earthworms (Eisenia fetida)

Abstract The purpose of this study was to investigate the effect of surface coating on the toxicity of silver nanoparticles (Ag NPs) soil. Earthworms (Eisenia fetida) were exposed to AgNO3 and Ag NPs with similar size ranges coated with either polyvinylpyrrolidone (hydrophilic) or oleic acid (amphiphilic) during a standard sub-chronic reproduction toxicity test. No significant effects on growth or mortality were observed within any of the test treatments. Significant decreases in reproduction were seen in earthworms exposed to AgNO3, (94.21 mg kg-1) as well as earthworms exposed to Ag NPs with either coating (727.6 mg kg-1 for oleic acid and 773.3 mg kg-1 for polyvinylpyrrolidone). The concentrations of Ag NPs at which effects were observed are much higher than predicted concentrations of Ag NPs in sewage sludge amended soils; however, the concentrations at which adverse effects of AgNO3 were observed are similar to the highest concentrations of Ag presently observed in sewage sludge in the United States. Earthworms accumulated Ag in a concentration-dependent manner from all Ag sources, with more Ag accumulating in tissues from AgNO3 compared to earthorms exposed to equivalent concentrations of Ag NPs. No differences were observed in Ag accumulation or toxicity between earthworms exposed to Ag NPs with polyvinylpyrrolidone or oleic acid coatings.

Evidence for Bioavailability of Au Nanoparticles from Soil and Biodistribution within Earthworms (Eisenia fetida)

Because Au nanoparticles (NPs) are resistant to oxidative dissolution and are easily detected, they have been used as stable probes for the behavior of nanomaterials within biological systems. Previous studies provide somewhat limited evidence for bioavailability of Au NPs in food webs, because the spatial distribution within tissues and the speciation of Au was not determined. In this study, we provide multiple lines of evidence, including orthogonal microspectroscopic techniques, as well as evidence from biological responses, that Au NPs are bioavailable from soil to a model detritivore (Eisenia fetida). We also present limited evidence that Au NPs may cause adverse effects on earthworm reproduction. This is perhaps the first study to demonstrate that Au NPs can be taken up by detritivores from soil and distributed among tissues. We found that primary particle size (20 or 55 nm) did not consistently influence accumulated concentrations on a mass concentration basis; however, on a particle number basis the 20 nm particles were more bioavailable. Differences in bioavailability between the treatments may have been explained by aggregation behavior in pore water. The results suggest that nanoparticles present in soil from activities such as biosolids application have the potential to enter terrestrial food webs.

Trophic Transfer of Au Nanoparticles from Soil along a Simulated Terrestrial Food Chain.

To determine if nanoparticles (NPs) could be transferred from soil media to invertebrates and then to secondary consumers, we examined the trophic transfer of Au NPs along a simulated terrestrial food chain. Earthworms (Eisenia fetida) were exposed to Au NPs in artificial soil media and fed to juvenile bullfrogs (Rana catesbeina). Earthworm Au concentrations were continuously monitored so that the cumulative dose to bullfrogs could be accurately estimated throughout the experiment. We exposed a second group of bullfrogs to equivalent doses of Au NPs by oral gavage to compare the bioavailability of NPs through direct exposure to trophic exposure. We observed accumulation of Au in liver, kidney, spleen, muscle, stomach, and intestine in both treatment groups. Tissue concentrations decreased on average of approximately 100-fold with each trophic-step. The total assimilated dose averaged only 0.09% of the administered dose for direct exposure (oral gavage), but 0.12% for the trophic exposure. The results suggest that manufactured NPs present in soil may be taken up into food chains and transferred to higher order consumers. They also suggest that Au NPs may be more bioavailable through trophic exposure than direct exposure and that trophic transfer may influence the biodistribution of particles once absorbed.

Influence of Natural Organic Matter and Surface Charge on the Toxicity and Bioaccumulation of Functionalized Ceria Nanoparticles in Caenorhabditis elegans

The objective of this study was to investigate the role of the CeO2 nanoparticle (NP) surface charge and the presence of natural organic matter (NOM) in determining bioavailability and toxicity to the model soil organism Caenorhabditis elegans. We synthesized CeO2-NPs functionalized with positively charged, negatively charged, and neutral coatings. The positively charged CeO2-NPs were significantly more toxic to C. elegans and bioaccumulated to a greater extent than the neutral and negatively charged CeO2-NPs. Surface charge also affected the oxidation state of Ce in C. elegans tissues after uptake. Greater reduction of Ce from Ce (IV) to Ce (III) was found in C. elegans, when exposed to the neutral and negatively charged relative to positively charged CeO2-NPs. The addition of humic acid (HA) to the exposure media significantly decreased the toxicity of CeO2-NPs, and the ratio of CeO2-NPs to HA influenced Ce bioaccumulation. When the concentration of HA was higher than the CeO2-NP concentration, Ce bioaccumulation decreased. These results suggest that the nature of the pristine coatings as a determinant of hazard may be greatly reduced once CeO2-NPs enter the environment and are coated with NOM.

Toxicogenomic responses of the model organism Caenorhabditis elegans to gold nanoparticles.

We used Au nanoparticles (Au-NPs) as a model for studying particle-specific effects of manufactured nanomaterials (MNMs) by examining the toxicogenomic responses in a model soil organism, Caenorhabditis elegans . Global genome expression for nematodes exposed to 4-nm citrate-coated Au-NPs at the LC(10) level (5.9 mg·L(-1)) revealed significant differential expression of 797 genes. The levels of expression for five genes (apl-1, dyn-1, act-5, abu-11, and hsp-4) were confirmed independently with qRT-PCR. Seven common biological pathways associated with 38 of these genes were identified. Up-regulation of 26 pqn/abu genes from noncanonical unfolded protein response (UPR) pathway and molecular chaperones (hsp-16.1, hsp-70, hsp-3, and hsp-4) were observed and are likely indicative of endoplasmic reticulum stress. Significant increase in sensitivity to Au-NPs in a mutant from noncanonical UPR (pqn-5) suggests possible involvement of the genes from this pathway in a protective mechanism against Au-NPs. Significant responses to Au-NPs in endocytosis mutants (chc-1 and rme-2) provide evidence for endocytosis pathway being induced by Au-NPs. These results demonstrate that Au-NPs are bioavailable and cause adverse effects to C. elegans by activating both general and specific biological pathways. The experiments with mutants further support involvement of several of these pathways in Au-NP toxicity and/or detoxification.

Nanomaterials in Biosolids Inhibit Nodulation, Shift Microbial Community Composition, and Result in Increased Metal Uptake Relative to Bulk/Dissolved Metals.

We examined the effects of amending soil with biosolids produced from a pilot-scale wastewater treatment plant containing a mixture of metal-based engineered nanomaterials (ENMs) on the growth of Medicago truncatula, its symbiosis with Sinorhizobium meliloti, and on soil microbial community structure. Treatments consisted of soils amended with biosolids generated with (1) Ag, ZnO, and TiO2 ENMs introduced into the influent wastewater (ENM biosolids), (2) AgNO3, Zn(SO4)2, and micron-sized TiO2 (dissolved/bulk metal biosolids) introduced into the influent wastewater stream, or (3) no metal added to influent wastewater (control). Soils were amended with biosolids to simulate 20 years of metal loading, which resulted in nominal metal concentrations of 1450, 100, and 2400 mg kg(-1) of Zn, Ag, and Ti, respectively, in the dissolved/bulk and ENM treatments. Tissue Zn concentrations were significantly higher in the plants grown in the ENM treatment (182 mg kg(-1)) compared to those from the bulk treatment (103 mg kg(-1)). Large reductions in nodulation frequency, plant growth, and significant shifts in soil microbial community composition were found for the ENM treatment compared to the bulk/dissolved metal treatment. These results suggest differences in metal bioavailability and toxicity between ENMs and bulk/dissolved metals at concentrations relevant to regulatory limits.

Toxicogenomic Responses of the Model Legume Medicago truncatula to Aged Biosolids Containing a Mixture of Nanomaterials (TiO2, Ag, and ZnO) from a Pilot Wastewater Treatment Plant

Toxicogenomic responses in Medicago truncatula A17 were monitored following exposure to biosolids-amended soils. Treatments included biosolids produced using a pilot wastewater treatment plant with either no metal introduced into the influent (control); bulk/ionic TiO2, ZnO, and AgNO3 added to influent (bulk/dissolved treatment); or Ag, ZnO, and TiO2 engineered nanomaterials added to influent (ENM treatment) and then added to soil, which was aged in the field for 6 months. In our companion study, we found inhibition of nodulation in the ENM but not in the bulk/dissolved treatment. Gene expression profiling revealed highly distinct profiles with more than 10-fold down-regulation in 239 genes in M. truncatula roots from the ENM treatment, while gene expression patterns were similar between bulk/dissolved and control treatments. In response to ENM exposure, many of the identified biological pathways, gene ontologies, and individual genes are associated with nitrogen metabolism, nodulation, metal homeostasis, and stress responses. Expression levels of nine genes were independently confirmed with qRT-PCR. Exposure to ENMs induced unique shifts in expression profiles and biological pathways compared with bulk/dissolved treatment, despite the lack of difference in bioavailable metal fractions, metal oxidation state, and coordination environment between ENM and bulk/dissolved biosolids. As populations of Sinorhizobium meliloti Rm2011 were similar in bulk/dissolved and ENM treatments, our results suggest that inhibition of nodulation in the ENM treatment was primarily due to phytotoxicity, likely caused by enhanced bioavailability of Zn ions.

Genetic and clonal diversity of two cattail species, Typha latifolia and T. angustifolia (Typhaceae), from Ukraine

Genetic and clonal diversity vary between two closely related cattail species (Typha angustifolia and T. latifolia) from Ukraine. This diversity was calculated from microsatellite data. Forty-eight percent of the total variation was partitioned between species, which formed distinct clusters in a dendrogram with no indication of hybrid populations. Typha angustifolia had higher heterozygosity at the species (H(es) = 0.66) and population (H(ep) = 0.49) levels than did T. latifolia (H(es) = 0.37 and H(ep) = 0.29, respectively). The higher number of alleles in T. angustifolia may be indicative of larger effective population sizes due to its higher seed production. Clonal diversity of T. angustifolia was lower than that of T. latifolia (N(g)/N(r) = 0.40 and 0.61, Simpsons D = 0.82 and 0.94, respectively). Correlations between clonal and genetic diversity were higher for T. latifolia than T. angustifolia, suggesting that the importance of factors and their interactions affecting this relationship are different for the two species. Latitudinal and longitudinal trends were not observed in either species despite the large sampling area. Population differentiation was relatively high with F(ST) of 0.24 and 0.29 for T. angustifolia and T. latifolia, respectively. Weak isolation by distance was observed for T. latifolia but not for T. angustifolia.

Multigenerational exposure to silver ions and silver nanoparticles reveals heightened sensitivity and epigenetic memory in Caenorhabditis elegans.

The effects from multigenerational exposures to engineered nanoparticles (ENPs) in their pristine and transformed states are currently unknown despite such exposures being an increasingly common scenario in natural environments. Here, we examine how exposure over 10 generations affects the sensitivity of the nematode Caenorhabditis elegans to pristine and sulfidized Ag ENPs and AgNO3. We also include populations that were initially exposed over six generations but kept unexposed for subsequent four generations to allow recovery from exposure. Toxicity of the different silver forms decreased in the order AgNO3, Ag ENPs and Ag2S ENPs. Continuous exposure to Ag ENPs and AgNO3 caused pronounced sensitization (approx. 10-fold) in the F2 generation, which was sustained until F10. This sensitization was less pronounced for Ag2S ENP exposures, indicating different toxicity mechanisms. Subtle changes in size and lifespan were also measured. In the recovery populations, the sensitivity to Ag ENPs and AgNO3 resulting from the initial multigenerational exposure persisted. Their response sensitivity for all endpoints was most closely related to the last ancestral exposed generation (F5), rather than unexposed controls. The mechanisms of transgenerational transfer of sensitivity are probably organized through the epigenome, and we encourage others to investigate such effects as a priority for mechanistic toxicology.