Lars Michael Skjolding

Aquatic Ecotoxicity Testing of Nanoparticles-The Quest To Disclose Nanoparticle Effects.

Abstract The number of products on the market containing engineered nanoparticles (ENPs) has increased significantly, and concerns have been raised regarding their ecotoxicological effects. Environmental safety assessments as well as relevant and reliable ecotoxicological data are required for the safe and sustainable use of ENPs. Although the number of publications on the ecotoxicological effects and uptake of ENPs is rapidly expanding, the applicability of the reported data for hazard assessment is questionable. A major knowledge gap is whether nanoparticle effects occur when test organisms are exposed to ENPs in aquatic test systems. Filling this gap is not straightforward, because of the broad range of ENPs and the different behavior of ENPs compared to “ordinary” (dissolved) chemicals in the ecotoxicity test systems. The risk of generating false negatives, and false positives, in the currently used tests is high, and in most cases difficult to assess. This Review outlines some of the pitfalls in the aquatic toxicity testing of ENPs which may lead to misinterpretation of test results. Response types are also proposed to reveal potential nanoparticle effects in the aquatic test organisms.

Chronic toxicity of silver nanoparticles to Daphnia magna under different feeding conditions.

Despite substantial information on the acute toxicity of silver nanoparticles (AgNP) to aquatic organisms, little is known about their potential chronic effects and the applicability of current test guidelines for testing nanomaterials. The purpose of this study was to study the influence of food availability on toxicity. This was done through a series of Daphnia magna 21-day reproduction tests (OECD 211) using 30 nm citric acid stabilized AgNP aimed at studying the influence of food abundance on the reproductive toxicity of AgNP in D. magna. The experiments were carried out as static renewal tests with exposure concentrations from 10 to 50 μg Ag/L, and test animals were fed green algae Pseudokirchneriella subcapitata in low and high food treatments. The endpoints recorded were survival, growth of parent animals and number of live neonates produced. Detrimental effects of AgNP on survival, growth and reproduction were observed in concentrations higher than 10 μg Ag/L, whereas the animals exposed to 10 μg Ag/L had larger body length and produced more offspring than controls at both food treatments. High food treatment resulted in higher animal survival, growth and reproduction compared to result found for low food treatment.

Trophic transfer of differently functionalized zinc oxide nanoparticles from crustaceans (Daphnia magna) to zebrafish (Danio rerio)

The potential uptake and trophic transfer of nanoparticles (NP) is not well understood so far and for ZnO NP the data presented in peer-reviewed literature is limited. In this paper the influence of surface functionalization on the uptake and depuration behavior of ZnO NP, ZnO-OH NP and ZnO-octyl NP in D. magna was studied. Bulk ZnO particles (≤5 μm) and ZnCl2 were used as references for uptake of particles and dissolved species of Zn, respectively. Furthermore, the trophic transfer of ZnO NP and ZnO-octyl NP from daphnids (Daphnia magna) to zebra fish (Danio rerio) was studied. For ZnO NP and ZnO-octyl NP fast uptakes in D. magna were observed, whereas no measurable uptake took place for ZnO-OH NP. Lower body burden of ZnCl2 was found compared to both ZnO NP and ZnO-octyl. Contrary, the body burden for bulk ZnO was higher than that of ZnO NP but lower than ZnO-octyl. The higher body burdens found for functionalized ZnO-octyl NP than for non-functionalized ZnO NP showed that that the functionalization of the NP has a high influence on the uptake and depuration behavior. Though no mortality was observed, the resulting body burdens were 9.6 times (ZnO NP) and 47 times (ZnO-octyl NP) higher than toxic levels reported for zinc in D. magna. Consequently, the zinc recovered in the animals was not solely due to soluble zinc, but agglomerates/aggregates of ZnO NP or ZnO-octyl NP contributed to the body burdens. The trophic transfer study showed uptake of both ZnO NP and ZnO-octyl NP reaching more than tenfold higher levels than those obtained through aqueous exposure in other studies. This study contributes to expand the available data on uptake behavior of differently functionalized ZnO NP in D. magna and the potential trophic transfer from zooplankton to fish.

Behavior and chronic toxicity of two differently stabilized silver nanoparticles to Daphnia magna.

While differences in silver nanoparticle (AgNP) colloidal stability, surface potential, or acute aquatic toxicity for differently stabilized AgNP have often been reported, these have rarely been studied in long-term ecotoxicity tests. In the current study, we investigated the chronic toxicity of AgNP to Daphnia magna over a 21-day period with two different stabilizers (citrate and detergent), representative for charge and sterical stabilizers, respectively. This was coupled with a series of short-term experiments, such as mass balance and uptake/depuration testing, to investigate the behavior of both types of AgNP during a typical media exchange period in the D. magna test for chronic toxicity. As expected, the sterically stabilized AgNP was more stable in the test medium, also in the presence of food; however, a higher uptake of silver after 24h exposure of the charge stabilized AgNP was found compared to the detergent-stabilized AgNP (0.046±0.006μgAgμgDW(-1) and 0.023±0.005μgAgμgDW(-1), respectively). In accordance with this, the higher reproductive effects and mortality were found for the charge-stabilized than for the sterically-stabilized silver nanoparticles in 21-d tests for chronic toxicity. LOEC was 19.2μgAgL(-1) for both endpoints for citrate-coated AgNP and >27.5μgAgL(-1) (highest tested concentration for detergent-stabilized AgNP). This indicates a link between uptake and toxicity. The inclusion of additional short-term experiments on uptake and depuration is recommended when longer-term chronic experiments with nanoparticles are conducted.

A critical analysis of the environmental dossiers from the OECD sponsorship programme for the testing of manufactured nanomaterials

In 2015, the OECD finally published the findings of its seven year testing programme for manufactured nanomaterials. Here, we present the first in-depth analysis of the published OECD dossiers with regards to data on physical and chemical properties, environmental fate and ecotoxicology. Each individual study in the dossiers was reviewed with regard to, among other, which OECD Test Guidelines (TG) were used, and the reliability assigned to the study. We furthermore analyzed in detail the suspension methods used, how media quality was quantified and physical and chemical characterization performed prior, during and/or at the end of the study. We find that the information in the dossiers present an incomplete portfolio of nanomaterial ecotoxicological evaluations that are difficult to draw substantive conclusions from and that most of the studies were not designed to investigate the validity of the OECD test guidelines. We acknowledge the effort of the OECD WPMN and recommend that a follow-on program is established with well-defined goals, end-points and direct funding to qualified research laboratories to ensure valid, rigorous, reproducible and efficient research.

An assessment of the importance of exposure routes to the uptake and internal localisation of fluorescent nanoparticles in zebrafish (Danio rerio), using light sheet microscopy

Abstract A major challenge in nanoecotoxicology is finding suitable methods to determine the uptake and localisation of nanoparticles on a whole-organism level. Some uptake methods have been associated with artefacts induced by sample preparation, including staining for electron microscopy. This study used light sheet microscopy (LSM) to define the uptake and localisation of fluorescently labelled nanoparticles in living organisms with minimal sample preparation. Zebrafish (Danio rerio) were exposed to fluorescent gold nanoparticles (Au NPs) and fluorescent polystyrene NPs via aqueous or dietary exposure. The in vivo uptake and localisation of NPs were investigated using LSM at different time points (1, 3 and 7 days). A time-dependent increase in fluorescence was observed in the gut after dietary exposure to both Au NPs and polystyrene NPs. No fluorescence was observed within gut epithelia regardless of the NP exposure route indicating no or limited uptake via intestinal villi. Fish exposed to polystyrene NPs through the aqueous phase emitted fluorescence signals from the gills and intestine. Fluorescence was also detected in the head region of the fish after aqueous exposure to polystyrene NPs. This was not observed for Au NPs. Aqueous exposure to Au NPs resulted in increased relative swimming distance, while no effect was observed for other exposures. This study supports that the route of exposure is essential for the uptake and subsequent localisation of nanoparticles in zebrafish. Furthermore, it demonstrates that the localisation of NPs in whole living organisms can be visualised in real-time, using LSM.

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 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.