Stakeholders' perspective on ecological modeling in environmental risk assessment of pesticides: challenges and opportunities.

The article closely examines the role of mechanistic effect models (e.g., population models) in the European environmental risk assessment (ERA) of pesticides. We studied perspectives of three stakeholder groups on population modeling in ERA of pesticides. Forty-three in-depth, semi-structured interviews were conducted with stakeholders from regulatory authorities, industry, and academia all over Europe. The key informant approach was employed in recruiting our participants. They were first identified as key stakeholders in the field and then sampled by means of a purposive sampling, where each stakeholder identified as important by others was interviewed and asked to suggest another potential participant for our study. Our results show that participants, although having different institutional backgrounds often presented similar perspectives and concerns about modeling. Analysis of repeating ideas and keywords revealed that all stakeholders had very high and often contradicting expectations from models. Still, all three groups expected effect models to become integrated in future ERA of pesticides. Main hopes associated with effect models were to reduce the amount of expensive and complex testing and field monitoring, both at the product development stage, and as an aid to develop mitigation measures. Our analysis suggests that, although the needs of stakeholders often overlapped, subtle differences and lack of trust hinder the process of introducing mechanistic effect models into ERA.

When and How to Conduct Ecotoxicological Tests Using Natural Field-Collected Sediment.

In recent years, the sediment compartment has gained more attention when performing toxicity tests, with a growing emphasis on gaining more ecological relevance in testing. Though many standard guidelines recommend using artificially formulated sediment, most sediment studies are using natural sediment collected in the field. Although the use of natural field-collected sediment contributes to more environmentally realistic exposure scenarios and higher well-being for sediment-dwelling organisms, it lowers comparability and reproducibility among studies as a result of, for example, differences in the base sediment depending on sampling site, background contamination, particle size distribution, or organic matter content. The aim of this methodology contribution is to present and discuss best practices related to collecting, handling, describing, and applying natural field-collected sediment in ecotoxicological testing. We propose six recommendations: (1) natural sediment should be collected at a well-studied site, historically and by laboratory analysis; (2) larger quantities of sediment should be collected and stored prior to initiation of an experiment to ensure a uniform sediment base; (3) any sediment used in ecotoxicological testing should be characterized, at the very least, for its water content, organic matter content, pH, and particle size distribution; (4) select spiking method, equilibration time, and experimental setup based on the properties of the contaminant and the research question; (5) include control-, treated similarly to the spiked sediment, and solvent control sediment when appropriate; and (6) quantify experimental exposure concentrations in the overlying water, porewater (if applicable), and bulk sediment at least at the beginning and the end of each experiment. Environ Toxicol Chem 2023;00:1-10. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Influence of copper treatment on bioaccumulation, survival, behavior, and fecundity in the fruit fly Drosophila melanogaster: Toxicity of copper oxide nanoparticles differ from dissolved copper.

Copper oxide (CuO) NPs are widely used and subsequently released into terrestrial ecosystems. In the present study, bioaccumulation and effects of CuO NPs and dissolved Cu was examined in the fruit fly Drosophila melanogaster after 7 and 10 days dietary exposure at concentrations ranging between 0.09 and 1.2 mg Cu ml-1 for dissolved Cu and between 0.2 and 11 mg Cu ml-1 for CuO NPs. Both Cu forms were bioaccumulated and affected survival and climbing in flies, but not egg-to-adult development. Dissolved Cu caused higher mortality than CuO NPs (CuO NPs 10-days LC50 was 2 times higher), whereas NPs affected climbing and decreased the number of eggs laid per female, potentially affecting fruit fly population size in terrestrial environments. Thus, the study indicates that CuO NPs might cause effects that are different from dissolved Cu due to differences in the mechanism of uptake or toxicity. Therefore, we need to consider relevant sublethal endpoints when assessing these CuO NPs to ensure that we do not overlook long-term effects.

Particles as carriers of matter in the aquatic environment: Challenges and ways ahead for transdisciplinary research.

A diverse array of natural and anthropogenic particles found in the aquatic environment, can act as carriers of co-transported matter (CTM), such as nutrients, genetic material and contaminants. Thus, understanding carrier particle transport will increase our understanding of local and global fluxes of exogenous CTM (affiliated with the particle) and endogenous CTM (an inherent part of the particle). In the present contribution, researchers from multiple disciplines collaborated to provide perspectives on the interactions between carrier particles and CTM, and the fundamentals of transport of particles found in the aquatic environment and the generic spherical smooth particles, often used to make predictions about particle behavior in suspension. Evidently, the particles in the aquatic environment show a great variety of characteristics and vary greatly from each other as well as from the generic particle. However, in spite of these differences, many fundamental concepts apply to particles in general. We emphasize the importance of understanding the basic concepts of transport of particle-associated CTM, and the main assumptions in the generic-founded models, which are challenged by the diverging characteristics of particles found in the aquatic environment, as paramount moving forward. Additionally, we identified the need for a conceptual and semantic link between different scientific fields of particle research and initiated the formation of a consistent terminology. Disciplinary and organizational (academic and funding) barriers need to be overcome to enable individual researchers to move beyond their knowledge sphere, to stimulate future interdisciplinary collaborations and to avoid research silos. Hereby, we can foster faster and better progress of evolving research fields on new and emerging anthropogenic carrier particles, and stimulate the development of solutions to the technological and environmental challenges.

Plastic nanoparticles cause mild inflammation, disrupt metabolic pathways, change the gut microbiota and affect reproduction in zebrafish: A full generation multi-omics study.

Plastic pollution has become a major concern on a global scale. The plastic is broken down into minuscule particles, which have an impact on the biosystems, however long-term impacts through an entire generation is largely unknown. Here, we present the first whole generation study exposing fish to a 500 nm polystyrene plastic particle at environmentally relevant concentrations. Short- and long-term adverse effects were investigated in the zebrafish model organism using a holistic multi-omics approach. The particles accumulated in the yolk sac of young larvae and short-term biological impacts included immune-relevant gene regulation related to inflammation and tolerance as well as disruption of metabolic processes, such as the fatty acid and lipid pathways. The long-term effects comprised gene regulations pointing towards skin and/or gill inflammation, dysbiosis of the gut microbiota, a tendency towards decreased condition factor in adult males as well as a lowered reproductive capability. From this study, it can be concluded that exposures to plastic nanoparticles have an impact on population as well as ecosystem level in fish and likely also in other vertebrates.

Biodynamics and adverse effects of CuO nanoparticles and CuCl2 in the oligochaete T. tubifex: Cu form influence biodynamics in water, but not sediment.

The use of copper oxide (CuO) NPs results in the release of these particles into the aquatic environment. Here, the particles settle out and accumulate in the sediment. However, little is known about the biodynamics of sediment-associated NPs in benthic organisms. We compared the toxicity and biodynamics of CuO NPs (7 nm) and dissolved Cu (CuCl2) in the sediment-dwelling oligochaete, Tubifex tubifex, to gain insights into the relative importance of metal form (CuCl2 vs CuO NPs) and exposure route (water vs sediment). Isotopically enriched 65Cu was used as a tracer to distinguish background from newly accumulated 65Cu in worms. For each exposure route, we conducted three experiments: one uptake, one elimination, and one longer-term net accumulation experiment to parameterize uptake and elimination of 65CuCl2 and 65CuO NPs in T. tubifex. 65Cu accumulation was detected for both 65CuCl2 and 65CuO NPs regardless of whether T. tubifex were exposed in sediment- or water-only setups. Water exposures to 65CuCl2 resulted in tail trauma whereas limited effects were seen for sediment exposures or exposures to 65CuO NPs via either exposure route. Uptake rate constants and accumulation of 65Cu in T. tubifex were higher following 65CuCl2 exposure than 65CuO NPs, in water, but not in sediment. Thus, the relative importance of exposure route and Cu form for uptake dynamics is not straightforward suggesting that findings on bioaccumulation and toxicity in water exposures cannot be directly extrapolated to sediment.

Influence of Aging on Bioaccumulation and Toxicity of Copper Oxide Nanoparticles and Dissolved Copper in the Sediment-Dwelling Oligochaete Tubifex tubifex: A Long-Term Study Using a Stable Copper Isotope.

For engineered metal nanoparticles (NPs), such as copper oxide (CuO) NPs, the sediment is recognized as a major compartment for NP accumulation. Sediment-dwelling organisms, such as the worm Tubifex tubifex, will be at particular risk of metal and metal NP exposure. However, a range of complex transformation processes in the sediment affects NP bioavailability and toxicity as the contamination ages. The objective of this study was to examine bioaccumulation and adverse effects of CuO NPs in T. tubifex compared to dissolved Cu (administered as CuCl2) and the influence of aging of spiked sediment. This was done in a 28-day exposure experiment with T. tubifex incubated in clean sediment or freshly spiked sediment with different concentrations of dissolved Cu (up to 230 µg g-1 dw) or CuO NPs (up to 40 µg g-1 dw). The experiment was repeated with the same sediments after it had been aged for 2 years. To obtain a distinct isotopic signature compared to background Cu, both Cu forms were based on the stable isotope 65Cu (>99%). The 28-day exposure to sediment-associated dissolved 65Cu and 65CuO NPs resulted in a clear concentration-dependent increase in the T. tubifex 65Cu body burden. However, despite the elevated 65Cu body burdens in exposed worms, limited adverse effects were observed in either of the two experiments (e.g., above 80% survival in all treatments, low or no effects on the growth rate, feeding rate, and reproduction). Organisms exposed to aged sediments had lower body burdens of 65Cu than those exposed to freshly spiked sediments and we suggest that aging decreases the bioavailability of both 65Cu forms. In this study, the use of a stable isotope made it possible to use environmentally realistic Cu concentrations and, at the same time, differentiate between newly accumulated 65Cu and background Cu in experimental samples despite the high background Cu concentrations in sediment and T. tubifex tissue. Realistic exposure concentrations and aging of NPs should preferably be included in future studies to increase environmental realism to accurately predict the environmental risk of metal NPs.

Dietary uptake and effects of copper in Sticklebacks at environmentally relevant exposures utilizing stable isotope-labeled 65CuCl2 and 65CuO NPs.

Copper oxide nanoparticles (CuO NPs) accumulating in sediment can be taken up by invertebrates that serve as prey for fish. Thus, it is likely that the latter are exposed to CuO NPs via the gut. However, to this day it is unknown if CuO NPs can be taken up via the gastrointestinal tract and if and in which tissues/organs they accumulate. To address this knowledge gap, we synthesized CuO NPs enriched in the stable isotope 65Cu and incorporated them at low concentration (5 µg 65Cu g-1 ww food) into a practical diet prepared from worm homogenate, which was then fed to Three-spined Stickleback (Gasterosteus aculeatus) for 16 days. For comparison, fish were exposed to a diet spiked with a 65CuCl2 solution. Background Cu and newly taken up 65Cu in fish tissues/organs including gill, stomach, intestine, liver, spleen, gonad and carcass and feces were quantified by ICP-MS. In addition, expression levels of genes encoding for proteins related to Cu uptake, detoxification and toxicity (ctr-1, gcl, gr, gpx, sod-1, cat, mta and zo-1) were measured in selected tissues using RT-qPCR. The obtained results showed that feces of fish fed 65CuO NP-spiked diet contained important amounts of 65Cu. Furthermore, there was no significant accumulation of 65Cu in any of the analyzed internal organs, though 65Cu levels were slightly elevated in liver. No significant modulation in gene expression was measured in fish exposed to 65CuO NP-spiked diet, except for metallothionein, which was significantly upregulated in intestinal tissue compared to control fish. Altogether, our results suggests that dietary absorption efficiency of CuO NPs, their uptake across the gastrointestinal barrier into the organism, and effects on Cu-related genes is limited at low, environmentally relevant exposure doses (0.2 µg 65Cu -1 fish ww day-1).

Effects of sediment-associated Cu on Tubifex tubifex - Insights gained by standard ecotoxicological and novel, but simple, bioturbation endpoints.

Sediments serve as both source and sink of contaminants (e.g., Cu) and biologically important materials (e.g., metals, nutrients). Bioturbation by benthic organisms is ecologically relevant as bioturbation affects the physio-chemical characteristics of sediments, thus altering nutrient and contaminant distribution and bioavailability. We examined the effects of sediment-associated Cu on T. tubifex with conventional toxicity endpoints, such as mortality and growth, and less commonly used non-destructive endpoints, such as bioturbation and feeding. An experimental approach was developed to examine the applicability of simple methods to detect effects on bioturbation and feeding. Two experiments were conducted with 7-day exposures to uncontaminated or Cu-spiked natural sediment at six Cu concentrations to examine Cu bioaccumulation and effects. Endpoints included worm mortality, feeding rate and growth (experiment A) and worm bioturbation (particle diffusion and maximum penetration depth, experiment B). A microparticle tracer was placed on the sediment surface and vertical particle transport was followed over time. Adverse effects were detected for all endpoints (bioturbation, feeding rate, growth and survival): a slight positive effect at the lowest Cu concentrations followed by adverse effects at higher concentrations indicating hormesis. These simple, non-destructive endpoints, provided valuable information and demonstrated that sediment-associated contaminants, such as Cu, can influence bioturbation activity, which in turn may affect the distribution of sediment-bound or particulate pollutants, such as the plastic microparticles studied here. Thus, we suggest to use simple endpoints, such as bioturbation and feeding rate, in ecotoxicity testing since these endpoint account for the influence of interactions between pollutants and benthos and, thus, increase ecological relevance.

Effects of copper oxide nanoparticles and copper ions to zebrafish (Danio rerio) cells, embryos and fry.

The use of engineered metal nanoparticles (NPs) is continuously increasing and so is the need for information regarding their toxicity. This study compares the toxicity of CuO NPs with ionic Cu in three zebrafish model systems; zebrafish hepatoma cell line (ZFL), fish embryo toxicity test (FET) and fry locomotion. In the ZFL tests, no significant cytotoxicity (cell death, decreased metabolic or cell membrane integrity) was detected for either treatment, though both significantly affected reactive oxygen species (ROS) production. Embryo mortality was affected by both Cu ions and CuO NPs with similar concentration-response relationships, whereas only Cu ions affected fry mortality (24h LC50≈30µM, ≈2mgCuL-1 for Cu ions and no significant mortality observed at up to 200µM, 12.7mgCuL-1 for CuO NP). Both Cu forms increased fry swimming activity during light cycles and decreased activity during dark cycles: Cu ions had significant impact at lower concentrations than CuO NPs. The implications are that Cu ions generally are more toxic than CuO NPs to embryos and fry but there is a marked difference in toxicity among the different zebrafish model systems. Metal NPs release into the environment may have adverse effects on fish and other aquatic organisms.

Not all that glitters is gold-Electron microscopy study on uptake of gold nanoparticles in Daphnia magna and related artifacts.

Increasing use of engineered nanoparticles has led to extensive research into their potential hazards to the environment and human health. Cellular uptake from the gut is sparsely investigated, and microscopy techniques applied for uptake studies can result in misinterpretations. Various microscopy techniques were used to investigate internalization of 10-nm gold nanoparticles in Daphnia magna gut lumen and gut epithelial cells following 24-h exposure and outline potential artifacts (i.e., high-contrast precipitates from sample preparation related to these techniques). Light sheet microscopy confirmed accumulation of gold nanoparticles in the gut lumen. Scanning transmission electron microscopy and elemental analysis revealed gold nanoparticles attached to the microvilli of gut cells. Interestingly, the peritrophic membrane appeared to act as a semipermeable barrier between the lumen and the gut epithelium, permitting only single particles through. Structures resembling nanoparticles were also observed inside gut cells. Elemental analysis could not verify these to be gold, and they were likely artifacts from the preparation, such as osmium and iron. Importantly, gold nanoparticles were found inside holocrine cells with disrupted membranes. Thus, false-positive observations of nanoparticle internalization may result from either preparation artifacts or mistaking disrupted cells for intact cells. These findings emphasize the importance of cell integrity and combining elemental analysis with the localization of internalized nanoparticles using transmission electron microscopy. Environ Toxicol Chem 2017;36:1503-1509. © 2016 SETAC.

Assessing and managing multiple risks in a changing world-The Roskilde recommendations.

Roskilde University (Denmark) hosted a November 2015 workshop, Environmental Risk-Assessing and Managing Multiple Risks in a Changing World. This Focus article presents the consensus recommendations of 30 attendees from 9 countries regarding implementation of a common currency (ecosystem services) for holistic environmental risk assessment and management; improvements to risk assessment and management in a complex, human-modified, and changing world; appropriate development of protection goals in a 2-stage process; dealing with societal issues; risk-management information needs; conducting risk assessment of risk management; and development of adaptive and flexible regulatory systems. The authors encourage both cross-disciplinary and interdisciplinary approaches to address their 10 recommendations: 1) adopt ecosystem services as a common currency for risk assessment and management; 2) consider cumulative stressors (chemical and nonchemical) and determine which dominate to best manage and restore ecosystem services; 3) fully integrate risk managers and communities of interest into the risk-assessment process; 4) fully integrate risk assessors and communities of interest into the risk-management process; 5) consider socioeconomics and increased transparency in both risk assessment and risk management; 6) recognize the ethical rights of humans and ecosystems to an adequate level of protection; 7) determine relevant reference conditions and the proper ecological context for assessments in human-modified systems; 8) assess risks and benefits to humans and the ecosystem and consider unintended consequences of management actions; 9) avoid excessive conservatism or possible underprotection resulting from sole reliance on binary, numerical benchmarks; and 10) develop adaptive risk-management and regulatory goals based on ranges of uncertainty. Environ Toxicol Chem 2017;36:7-16. © 2016 SETAC.

Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part II: Subcellular distribution following sediment exposure.

The use and likely incidental release of metal nanoparticles (NPs) is steadily increasing. Despite the increasing amount of published literature on metal NP toxicity in the aquatic environment, very little is known about the biological fate of NPs after sediment exposures. Here, we compare the bioavailability and subcellular distribution of copper oxide (CuO) NPs and aqueous Cu (Cu-Aq) in the sediment-dwelling worm Lumbriculus variegatus. Ten days (d) sediment exposure resulted in marginal Cu bioaccumulation in L. variegatus for both forms of Cu. Bioaccumulation was detected because isotopically enriched 65Cu was used as a tracer. Neither burrowing behavior or survival was affected by the exposure. Once incorporated into tissue, Cu loss was negligible over 10 d of elimination in clean sediment (Cu elimination rate constants were not different from zero). With the exception of day 10, differences in bioaccumulation and subcellular distribution between Cu forms were either not detectable or marginal. After 10 d of exposure to Cu-Aq, the accumulated Cu was primarily partitioned in the subcellular fraction containing metallothionein-like proteins (MTLP, ≈40%) and cellular debris (CD, ≈30%). Cu concentrations in these fractions were significantly higher than in controls. For worms exposed to CuO NPs for 10 d, most of the accumulated Cu was partitioned in the CD fraction (≈40%), which was the only subcellular fraction where the Cu concentration was significantly higher than for the control group. Our results indicate that L. variegatus handle the two Cu forms differently. However, longer-term exposures are suggested in order to clearly highlight differences in the subcellular distribution of these two Cu forms.

Bioaccumulation, subcellular distribution and toxicity of sediment-associated copper in the ragworm Nereis diversicolor: The relative importance of aqueous copper, copper oxide nanoparticles and microparticles.

The sediment-dwelling ragworm, Nereis diversicolor was exposed to sediment spiked with aqueous Cu (CuAq, CuCl2), CuO nanoparticles (CuONP) or CuO microparticles (CuOMicro) at 150 µg Cu g(-1) dw sediment for 10d. Exposures to CuAq and CuOMicro caused mortality (62.5 and 37.5%, respectively), whereas mean burrowing time increased during exposure to CuAq and CuONP from 0.12 h (controls) to 19.3 and 12.2 h, respectively. All Cu treatments bioaccumulated, especially CuAq (up to 4 times more than the other treatments). Cu was roughly equally distributed among the five subcellular fractions in controls and worms exposed to CuONP or CuOMicro. In contrast, ≈50% of accumulated Cu in CuAq exposed worms was found in metal rich granules and significantly more Cu was present in heat-denatured proteins and organelles than in worms exposed to CuOMicro or in controls. Our results suggest that Cu form affects its bioaccumulation and subsequent toxicity and detoxification in a polychaete like N. diversicolor.

Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells.

CuO NPs have previously been reported as toxic to a range of cell cultures including kidney epithelial cells from the frog, Xenopus laevis (A6). Here we examine the molecular mechanisms affecting toxicity of Cu in different forms and particle sizes. A6 cells were exposed to ionic Cu (Cu2+) or CuO particles of three different sizes: CuO NPs of 6 nm (NP6), larger Poly-dispersed CuO NPs of <100 nm (Poly) and CuO Micro particles of <5 µm (Micro), at 200 µM, equal to 12.7 mg Cu/L. Poly was significantly more toxic than NP6, Micro and Cu2+ to A6 cells, causing DNA damage, decreased cell viability and levels of reduced glutathione (GSH) and eventually cell death. We show that ROS (Reactive Oxygen Species) generation plays a key role and occurs early in Poly toxicity as Poly-induced DNA damage and cell death could be mitigated by the antioxidant NAC (N-acetyl-cysteine). Here we propose a model of the sequence of events explaining Poly toxicity. Briefly, the events include: cellular uptake, most likely via endocytosis, production of ROS, which cause DNA damage that activates a signaling pathway which eventually leads to cell death, mainly via apoptosis.

Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part I: Relative importance of water and sediment as exposure routes.

Copper oxide (CuO) nanoparticles (NPs) are widely used, and likely released into the aquatic environment. Both aqueous (i.e., dissolved Cu) and particulate Cu can be taken up by organisms. However, how exposure routes influence the bioavailability and subsequent toxicity of Cu remains largely unknown. Here, we assess the importance of exposure routes (water and sediment) and Cu forms (aqueous and nanoparticulate) on Cu bioavailability and toxicity to the freshwater oligochaete, Lumbriculus variegatus, a head-down deposit-feeder. We characterize the bioaccumulation dynamics of Cu in L. variegatus across a range of exposure concentrations, covering both realistic and worst-case levels of Cu contamination in the environment. Both aqueous Cu (Cu-Aq; administered as Cu(NO3)2) and nanoparticulate Cu (CuO NPs), whether dispersed in artificial moderately hard freshwater or mixed into sediment, were weakly accumulated by L. variegatus. Once incorporated into tissues, Cu elimination was negligible, i.e., elimination rate constants were in general not different from zero for either exposure route or either Cu form. Toxicity was only observed after waterborne exposure to Cu-Aq at very high concentration (305µgL(-1)), where all worms died. There was no relationship between exposure route, Cu form or Cu exposure concentration on either worm survival or growth. Slow feeding rates and low Cu assimilation efficiency (approximately 30%) characterized the uptake of Cu from the sediment for both Cu forms. In nature, L. variegatus is potentially exposed to Cu via both water and sediment. However, sediment progressively becomes the predominant exposure route for Cu in L. variegatus as Cu partitioning to sediment increases.

Influence of copper oxide nanoparticle shape on bioaccumulation, cellular internalization and effects in the estuarine sediment-dwelling polychaete, Nereis diversicolor.

CuO nanoparticles (NPs) released into the aquatic environment will likely accumulate in the sediment. Here we synthesized and characterized CuO NPs with different shapes and thus sizes: spheres, rods and spindles. Nereis diversicolor were exposed for 10 days to control sediment or sediment spiked with CuO NPs or aqueous Cu (Cu-Aq, CuCl2) at 7, 70 and 140 µg Cu g(-1) dw sediment. Cu from all Cu treatments accumulated in worms in a concentration-dependent manner. Only Cu-Aq decreased burrowing, suggesting that worms avoid Cu when added to sediment as Cu-Aq, but not CuO NPs. Transmission Electron Microscopy of gut sections indicated limited presence of CuO NP-like objects in the gut lumen, but evidence on whether accumulated Cu from CuO NP exposure was internalized as particles was not conclusive. Overall, bioavailability and avoidance was not influenced by particle shape or size, whereas Cu form (Cu-Aq vs particulate) and exposure concentration had significant impact.

Toxicity of CuO nanoparticles and Cu ions to tight epithelial cells from Xenopus laevis (A6): effects on proliferation, cell cycle progression and cell death.

Nanoparticles (NPs) have unique chemical and physical properties caused by their small size (1-100 nm) and high surface to volume ratio. This means that the NPs are potentially more toxic than their bulk counterparts. In the present study a cultured epithelial cell line from Xenopus laevis (A6) was used to investigate toxicity of copper (Cu) in 3 different forms; Cu ions (Cu(2+)), CuO NPs (6 nm) and poly-dispersed CuO NPs (100 nm, poly-CuO). Continuous exposures at concentrations of 143-200 µM demonstrated that cytotoxicity differed among the 3 Cu forms tested and that the effects depend on cell state (dividing or differentiated). Dividing cells treated with poly-CuO, CuO NPs (6 nm) or Cu(2+) showed cell cycle arrest and caused significant increase in cell death via apoptosis after 48 h, 6 and 7 days of treatment, respectively. Treatment with either CuO NPs (6 nm) or Cu(2+) caused significant decrease in cell proliferation. Treatments of differentiated cells, revealed the same patterns of toxicity for Cu forms tested, but after shorter exposure periods.