Amalie Thit

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.

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.

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.

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.

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.

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.

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.

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.

Acute toxicity of copper oxide nanoparticles to Daphnia magna under different test conditions

ABSTRACT The acute toxicity of monodispersed 6 nm and <100 nm poly-dispersed copper oxide nanoparticles toward Daphnia magna was assessed using 48 h immobilization tests. CuSO4 was used as a reference. Four different exposure conditions were tested, to study whether the toxicity of the nanoparticle suspensions changed in a way similar to what is known for dissolved Cu: first in ISO standard test conditions (pH 7.8), second with slight acidity (pH 6.5), third in the presence of citric acid, and fourth in the presence of humic acid. For all four exposure conditions, the toxicity of Cu employed in the three forms followed the same sequence, i.e., CuSO4 > monodispersed 6 nm CuO ≫ poly-dispersed CuO. The toxicity of all Cu forms decreased from pH 6.5, ≫ pH 7.8, > pH 7.8 + citric acid, to ≫ pH 7.8 + humic acid. This pattern is in agreement with concentrations of Cu2+ calculated using the equilibrium model MINTEQ. These findings show that the acute toxicity of copper oxide nanoparticles is governed by test water composition and the chemical species Cu2+.

Not all that glitters is gold-Electron microscopy study on uptake of gold nanoparticles in Daphnia Magna and Related Artefacts

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.