Pristine and Sulfidized Zinc Oxide Nanoparticles Promote the Release and Decomposition of Organic Carbon in the Legume Rhizosphere.

The effects and mechanisms of zinc oxide nanoparticles (ZnO NPs) and their aging products, sulfidized (s-) ZnO NPs, on the carbon cycling in the legume rhizosphere are still unclear. We observed that, after 30 days of cultivation, in the rhizosphere soil of Medicago truncatula, under ZnO NP and s-ZnO NP treatments, the dissolved organic carbon (DOC) concentrations were significantly increased by 1.8- to 2.4-fold compared to Zn2+ treatments, although the soil organic matter (SOM) contents did not change significantly. Compared to Zn2+ additions, the additions of NPs significantly induced the production of root metabolites such as carboxylic acids and amino acids and also stimulated the growth of microbes involved in the degradations of plant-derived and recalcitrant SOM, such as bacteria genera RB41 and Bryobacter, and fungi genus Conocybe. The bacterial co-occurrence networks indicated that microbes associated with SOM formation and decomposition were significantly increased under NP treatments. The adsorption of NPs by roots, the generation of root metabolites (e.g., carboxylic acid and amino acid), and enrichment of key taxa (e.g., RB41 and Gaiella) were the major mechanisms by which ZnO NPs and s-ZnO NPs drove DOC release and SOM decomposition in the rhizosphere. These results provide new perspectives on the effect of ZnO NPs on agroecosystem functions in soil-plant systems.

Bioanalytical approaches for the detection, characterization, and risk assessment of micro/nanoplastics in agriculture and food systems.

This review discusses the most recent literature (mostly since 2019) on the presence and impact of microplastics (MPs, particle size of 1 µm to 5 mm) and nanoplastics (NPs, particle size of 1 to 1000 nm) throughout the agricultural and food supply chain, focusing on the methods and technologies for the detection and characterization of these materials at key entry points. Methods for the detection of M/NPs include electron and atomic force microscopy, vibrational spectroscopy (FTIR and Raman), hyperspectral (bright field and dark field) and fluorescence imaging, and pyrolysis-gas chromatography coupled to mass spectrometry. Microfluidic biosensors and risk assessment assays of MP/NP for in vitro, in vivo, and in silico models have also been used. Advantages and limitations of each method or approach in specific application scenarios are discussed to highlight the scientific and technological obstacles to be overcome in future research. Although progress in recent years has increased our understanding of the mechanisms and the extent to which MP/NP affects health and the environment, many challenges remain largely due to the lack of standardized and reliable detection and characterization methods. Most of the methods available today are low-throughput, which limits their practical application to food and agricultural samples. Development of rapid and high-throughput field-deployable methods for onsite screening of MP/NPs is therefore a high priority. Based on the current literature, we conclude that detecting the presence and understanding the impact of MP/NP throughout the agricultural and food supply chain require the development of novel deployable analytical methods and sensors, the combination of high-precision lab analysis with rapid onsite screening, and a data hub(s) that hosts and curates data for future analysis.

Uptake and Bioactivity of Chitosan/Double-Stranded RNA Polyplex Nanoparticles in Caenorhabditis elegans.

In this study, we investigated chitosan/dsRNA polyplex nanoparticles as RNAi agents in the nematode Caenorhabditis elegans. By measurement of an easily observed phenotype and uptake of fluorescently labeled dsRNA, we demonstrate that chitosan/dsRNA polyplex nanoparticles are considerably more effective at gene knockdown on a whole body concentration basis than naked dsRNA. Further, we show that chitosan/dsRNA polyplex nanoparticles introduce dsRNA into cells via a different mechanism than the canonical sid-1 and sid-2 pathway. Clathrin-mediated endocytosis is likely the main uptake mechanism. Finally, although largely reported as nontoxic, we have found that chitosan, as either polyplex nanoparticles or alone, is capable of downregulating the expression of myosin. Myosin is a critical component of growth and development in eukaryotes, and we have observed reductions in both growth rate and reproduction in chitosan exposed C. elegans. Given the increased potency, noncanonical uptake, and off-target effects that we identified, these findings highlight the need for a rigorous safety assessment of nano-RNAi products prior to deployment. Specifically, the potential adverse effects of the nanocarrier and its components need to be considered.

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.

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.

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.

A genetic map of Peromyscus with chromosomal assignment of linkage groups (a Peromyscus genetic map).

The rodent genus Peromyscus is the most numerous and species-rich mammalian group in North America. The naturally occurring diversity within this genus allows opportunities to investigate the genetic basis of adaptation, monogamy, behavioral and physiological phenotypes, growth control, genomic imprinting, and disease processes. Increased genomic resources including a high quality genetic map are needed to capitalize on these opportunities. We produced interspecific hybrids between the prairie deer mouse (P. maniculatus bairdii) and the oldfield mouse (P. polionotus) and scored meiotic recombination events in backcross progeny. A genetic map was constructed by genotyping of backcross progeny at 185 gene-based and 155 microsatellite markers representing all autosomes and the X-chromosome. Comparison of the constructed genetic map with the molecular maps of Mus and Rattus and consideration of previous results from interspecific reciprocal whole chromosome painting allowed most linkage groups to be unambiguously assigned to specific Peromyscus chromosomes. Based on genomic comparisons, this Peromyscus genetic map covers ~83% of the Rattus genome and 79% of the Mus genome. This map supports previous results that the Peromyscus genome is more similar to Rattus than Mus. For example, coverage of the 20 Rattus autosomes and the X-chromosome is accomplished with only 28 segments of the Peromyscus map, but coverage of the 19 Mus autosomes and the X-chromosome requires 40 chromosomal segments of the Peromyscus map. Furthermore, a single Peromyscus linkage group corresponds to about 91% of the rat and only 76% of the mouse X-chromosomes.

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.

Effects of Multiple Stressors, Pristine or Sulfidized Silver Nanomaterials, and a Pathogen on a Model Soil Nematode Caenorhabditis elegans.

Previous research using the model soil nematode Caenorhabditis elegans has revealed that silver nanoparticles (AgNP) and their transformed counterpart, sulfidized AgNP (sAgNP), reduce their reproduction and survival. To expand our understanding of the environmental consequences of released NP, we examined the synergistic/antagonistic effects of AgNP and sAgNP along with AgNO3 (ionic control) on C. elegans infected with the pathogen Klebsiella pneumoniae. Individual exposures to each stressor significantly decreased nematode reproduction compared to controls. Combined exposures to equitoxic EC30 concentrations of two stressors, Ag in nanoparticulate (AgNP or sAgNP) or ionic form and the pathogen K. pneumoniae, showed a decline in the reproduction that was not significantly different compared to individual exposures of each of the stressors. The lack of enhanced toxicity after simultaneous combined exposure is partially due to Ag decreasing K. pneumoniae pathogenicity by inhibiting biofilm production outside the nematode and significantly reducing viable pathogens inside the host. Taken together, our results indicate that by hindering the ability of K. pneumoniae to colonize the nematode's intestine, Ag reduces K. pneumoniae pathogenicity regardless of Ag form. These results differ from our previous research where simultaneous exposure to zinc oxide (ZnO) NP and K. pneumoniae led to a reproduction level that was not significantly different from the controls.

Metal-Organic Framework-Enabled Sustainable Agrotechnologies: An Overview of Fundamentals and Agricultural Applications.

With aggravated abiotic and biotic stresses from increasing climate change, metal-organic frameworks (MOFs) have emerged as versatile toolboxes for developing environmentally friendly agrotechnologies aligned with agricultural practices and safety. Herein, we have explored MOF-based agrotechnologies, focusing on their intrinsic properties, such as structural and catalytic characteristics. Briefly, MOFs possess a sponge-like porous structure that can be easily stimulated by the external environment, facilitating the controlled release of agrochemicals, thus enabling precise delivery of agrochemicals. Additionally, MOFs offer the ability to remove or degrade certain pollutants by capturing them within their pores, facilitating the development of MOF-based remediation technologies for agricultural environments. Furthermore, the metal-organic hybrid nature of MOFs grants them abundant catalytic activities, encompassing photocatalysis, enzyme-mimicking catalysis, and electrocatalysis, allowing for the integration of MOFs into degradation and sensing agrotechnologies. Finally, the future challenges that MOFs face in agrotechnologies were proposed to promote the development of sustainable agriculture practices.

Multiple stressor effects on a model soil nematode, Caenorhabditis elegans: Combined effects of the pathogen Klebsiella pneumoniae and zinc oxide nanoparticles.

Research utilizing the model soil nematode Caenorhabditis elegans has revealed that agriculturally relevant nanoparticles (NP), such as zinc oxide NP (ZnONP), cause toxicity at low concentrations and disrupt molecular pathways of pathogen resistance. However, in most nanotoxicity assessments, model organisms are exposed to a single stressor but in nature organisms are affected by multiple sources of stress, including infections, which might exacerbate or mitigate negative effects of NP exposure. Thus, to expand our understanding of the environmental consequences of released NP, this project examined the synergistic/antagonistic effects of ZnONP on C. elegans infected with a common pathogen, Klebsiella pneumoniae. Individual exposures of C. elegans to ZnONP, zinc sulfate (Zn2+ ions) or K. pneumoniae significantly decreased nematode reproduction compared to controls. To assess the combined stress of ZnONP and K. pneumoniae, C. elegans were exposed to equitoxic EC30 concentrations of ZnONP (or Zn ions) and K. pneumoniae. After the combined exposure there was no decrease in reproduction. This complete elimination of reproductive toxicity was unexpected because exposures were conducted at EC30 Zn concentrations and reproductive toxicity due to Zn should have occurred. Amelioration of the pathogen effects by Zn are partially explained by the Zn impact on the K. pneumoniae biofilm. Quantitative assessments showed that external biofilm production and estimated colony forming units (CFU) of K. pneumoniae within the nematodes were significantly decreased. Taken together, our results suggest that during the combined exposure of C. elegans to both stressors Zn in ionic or particulate form inhibits K. pneumoniae ability to colonize nematode's intestine through decreasing pathogen biofilm formation. This highlights the unpredictable nature of combined stressor effects, calling into question the utility of exposures in simplified laboratory media.

Silver contamination and its toxicity and risk management in terrestrial and aquatic ecosystems.

Silver (Ag), a naturally occurring, rare and precious metal, is found in major minerals such as cerargyrite (AgCl), pyrargyrite (Ag3SbS3), proustite (Ag3AsS3), and stephanite (Ag5SbS4). From these minerals, Ag is released into soil and water through the weathering of rocks and mining activities. Silver also enters the environment by manufacturing and using Ag compounds in electroplating and photography, catalysts, medical devices, and batteries. With >400 t of Ag NPs produced yearly, Ag NPs have become a rapidly growing source of anthropogenic Ag input in the environment. In soils and natural waters, most Ag is sorbed to soil particles and sediments and precipitated as oxides, carbonates, sulphides, chlorides and hydroxides. Silver and its compounds are toxic, and humans and other animals are exposed to Ag through inhalation of air and the consumption of Ag-contaminated food and drinking water. Remediation of Ag-contaminated soil and water sources can be achieved through immobilization and mobilization processes. Immobilization of Ag in soil and groundwater reduces the bioavailability and mobility of Ag, while mobilization of Ag in the soil can facilitate its removal. This review provides an overview of the current understanding of the sources, geochemistry, health hazards, remediation practices and regulatory mandates of Ag contamination in complex environmental settings, including soil and aquatic ecosystems. Knowledge gaps and future research priorities in the sustainable management of Ag contamination in these settings are also discussed.

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.

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.

Potential Environmental and Health Implications from the Scaled-Up Production and Disposal of Nanomaterials Used in Biosensors.

Biosensors often combine biological recognition elements with nanomaterials of varying compositions and dimensions to facilitate or enhance the operating mechanism of the device. While incorporating nanomaterials is beneficial to developing high-performance biosensors, at the stages of scale-up and disposal, it may lead to the unmanaged release of toxic nanomaterials. Here we attempt to foster connections between the domains of biosensors development and human and environmental toxicology to encourage a holistic approach to the development and scale-up of biosensors. We begin by exploring the toxicity of nanomaterials commonly used in biosensor design. From our analysis, we introduce five factors with a role in nanotoxicity that should be considered at the biosensor development stages to better manage toxicity. Finally, we contextualize the discussion by presenting the relevant stages and routes of exposure in the biosensor life cycle. Our review found little consensus on how the factors presented govern nanomaterial toxicity, especially in composite and alloyed nanomaterials. To bridge the current gap in understanding and mitigate the risks of uncontrolled nanomaterial release, we advocate for greater collaboration through a precautionary One Health approach to future development and a movement towards a circular approach to biosensor use and disposal.

Evidence for avoidance of Ag nanoparticles by earthworms (Eisenia fetida).

Silver nanoparticles have been incorporated into a wide variety of consumer products, ideally acting as antimicrobial agents. Silver exposure has long been known to cause toxic effects to a wide variety of organisms, making large scale production of silver nanoparticles a potential hazard to environmental systems. Here we describe the first evidence that an organism may be able to sense manufactured nanoparticles in a complex, environmentally relevant exposure and that the presence of nanoparticles alters the organism's behavior. We found that earthworms (Eisenia fetida) consistently avoid soils containing silver nanoparticles and AgNO(3) at similar concentrations of Ag. However, avoidance of silver nanoparticles occurred over 48 h, while avoidance of AgNO(3) was immediate. It was determined that avoidance of silver nanoparticles could not be explained by release of silver ions or any changes in microbial communities caused by the introduction of Ag. This leads us to conclude that the earthworms were in some way sensing the presence of nanoparticles over the course of a 48 h exposure and choosing to avoid exposure to them. Our results demonstrate that nanoparticle interactions with organisms may be unpredictable and that these interactions may result in ecologically significant effects on behavior at environmentally relevant concentrations.

Responses of soil bacteria and fungal communities to pristine and sulfidized zinc oxide nanoparticles relative to Zn ions.

Zinc oxide nanoparticles (ZnO NPs) are attracting much interest due to their potential toxicity and ubiquity in consumer products. However, understanding of pristine and transformed ZnO NPs impact on soil microbial communities is still limited. Here, we explored changes in the microbial communities of soils treated with pristine and sulfidized ZnO NPs (s-ZnO NPs), and their corresponding Zn ions (ZnSO4) for 30 and 90 days exposures at 100 and 500 mg Zn kg-1. The similarity in bacterial community responses was observed between ZnO NPs and s-ZnO NPs, and these Zn treatments significantly affected the bacterial communities at 90 days, which exhibited distinct patterns compared to ZnSO4. The single-time tested DTPA and H2O extractable Zn ions could not fully explain the observed ZnO NPs and s-ZnO NPs impact on bacterial communities. The two most dominant phylum Nitrospirae and Actinobacteria, associated with the reduction of NH4+-N and dissolved organic carbon, demonstrated significant changes in soils exposed to ZnO NPs and s-ZnO NPs. This suggests the potential long-term impact of transformed ZnO NPs on soil carbon and nitrogen cycling. For fungal communities, we did not find the distinct response patterns of fungal communities between nanoparticulate and ionic Zn exposures.

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.

Five Hundred Microsatellite Loci for Peromyscus.

Mice of the genus Peromyscus, including several endangered subspecies, occur throughout North America and have been important models for conservation research. We describe 526 primer pairs that amplify microsatellite DNA loci for P. maniculatus bairdii, 467 of which also amplify in P. polionotus subgriseus. For 12 of these loci, we report diversity data from a natural population. These markers will be an important resource for future genomic studies of Peromyscus evolution and mammalian conservation.

Effects of particle size on chemical speciation and bioavailability of copper to earthworms (Eisenia fetida) exposed to copper nanoparticles.

To investigate the role of particle size on the oxidation, bioavailability, and adverse effects of manufactured Cu nanoparticles (NPs) in soils, we exposed the earthworm Eisenia ferida to a series of concentrations of commercially produced NPs labeled as 20- to 40-nm or < 100-nm Cu in artificial soil media. Effects on growth, mortality, reproduction, and expression of a variety of genes associated with metal homeostasis, general stress, and oxidative stress were measured. We also used X-ray absorption spectroscopy and scanning X-ray fluorescence microscopy to characterize changes in chemical speciation and spatial distribution of the NPs in soil media and earthworm tissues. Exposure concentrations of Cu NPs up to 65 mg kg(-1) caused no adverse effects on ecologically relevant endpoints. Increases in metallothionein expression occurred at concentrations exceeding 20 mg kg(-1) of Cu NPs and concentrations exceeding 10 mg kg(-1) of CuSO4. Based on the relationship of Cu tissue concentration to metallothionein expression level and the spatial distribution and chemical speciation of Cu in the tissues, we conclude that Cu ions and oxidized Cu NPs were taken up by the earthworms. This study suggests that oxidized Cu NPs may enter food chains from soil but that adverse effects in earthworms are likely to occur only at relatively high concentrations (> 65 mg Cu kg(-1) soil).