Nanomaterials in Environmental Samples
Sensitive Method for the Detection in the ppb-Range
- Fig. 1: Detail of a transmission electron microscopic (TEM) image of a human adipogenic differentiated cell: untreated (without nanoparticles) at the top, treated with gold nanoparticles at the bottom. The particles accumulate in the lipid droplets of the cell.
- Fig. 2: Fluorescence microscopic image of an epithelial cell after exposure to iron oxide nanoparticles. Green: cell membrane. Red: iron oxide nanoparticles accumulated inside the cell.
In textiles, cosmetics, food and food supplements, washing agents or as coatings of kitchenware nanomaterials (NM) are processed. The numerous potential applications and the continuously rising volume of synthetic NM illustrate the increasing exposure of human and environment with synthetic NM. Due to a lack in measurement technologies for determining the continuity and intensity of NM exposure of humans, concrete data is missing for assessing the NM exposure and the risk potential.
Knowing the real concentration of NM occurring in the environment is essential for assessing the dimension of hazard for environmental organisms. So far, no studies exist determining the real NM concentrate on in different environmental compartments along the food chain. Up to now, the entry and the distribution of the NM in the environment have been simulated only via computer modelling. The exact concentration of synthetic NM in the environment, their functionality and accumulation behavior as well as their effect on the environmental system is unclear now.
Within the framework of the project NanoUmwelt a sensitive method for testing a variety of environmental samples such as river water, animal tissue, or human urine and blood that can detect NM at a concentration level of nanogram per liter (ppb – parts per billion) has been successfully developed. Using the new method, it is now possible to detect not just large amounts of NM in clear fluids, as was previously the case, but also very few particles in complex substance mixtures such as human blood or soil samples. The approach is based on field-flow fractionation (FFF), which can be used to separate complex heterogeneous mixtures of fluids and particles into their component parts – while simultaneously sorting the key components by size. This is achieved by the combination of a controlled flow of fluid and a physical separation field, which acts perpendicularly on the flowing suspension. In a first step the project team separates the NM via asymmetric flow-field-flow-fractionation (AF4) (AF2000 MultiFlow FFF, Postnova Analytics GmbH) according to material type and/or size. In a second step follows the analysis of the separated particle fractions using different detectors, as e.g.
MALS-, UV- or DLS detector. Furthermore, the surface charge (ZetaSizer Nano) and the material will be determined via ICP-MS.
For the detection process to work, the environmental samples have to be appropriately processed. The environmental and human samples, as river water, human urine, human blood and fish tissue were prepared with special enzymes to be ready for AF4 analysis. During process development, it is necessary to make sure that the NM are not destroyed or modified during the digestion process, allowing detecting the real characteristics of the NM in the environmental and human samples.
Nanomaterials in the Food Chain
The aim is the first-time determination of the real NM concentrations in different environmental compartments along the food chain. Inter alia the essential question “How does the transfer of NM takes place along the food chain?“ is addressed. In a „small“ food chain comprising daphnia and zebra danios, the transfer of NM to zebra dinos after feeding them with TiO2-exposed daphnia, has already been shown . In addition, correlations of a high NM exposure and an increased accumulation of heavy metals, as e.g. cadmium or arsenic, in waters are also documented .
The physico-chemical properties of the NM during the NM life cycle have been studied insufficiently until now. Therefore, not only water samples were collected, but also samples of primary and secondary consumers with key functions in the food chain and the real concentration and characteristics of the NM in these samples were determined.
Nanomaterials in Human Tissue Samples
Fraunhofer IBMT has been running the “German Environmental Specimen Bank-Human Samples (ESB)” since January 2012 on behalf of Germany’s Environment Agency (UBA). Each year the working group on Biomonitoring & Cryobanks, collects blood and urine samples from 120 volunteers in four cities in Germany. Individual samples are a valuable tool for mapping the trends over time of human exposure to pollutants. In addition, blood and urine samples have been donated for the NanoUmwelt project, put into cryostorage at Fraunhofer IBMT and used to develop the new detection method. After approval by the UBA, some of the human samples in the ESB archive can also be examined using the new method.
Effects of Nanomaterials in the Low-dose Range
Besides the analytics of the NM in environmental- and human samples, also human toxicological effects of NM in the low-dose range were investigated. On cellular level NM exposure could trigger different effects. Besides inflammatory mechanisms, changes in proliferation, motility and adhesion could be induced and could lead to apoptosis, necrosis or autophagocytosis. In the nucleus NM could cause DNA damage or chromosomal aberration.
The barrier mobility presents an additional parameter for the evaluation of the hazard potential of NM to human beings and the environment. At this, the focus has been on the investigation of NM transport over biological barriers, as the lung or the gastrointestinal tract. It distinguishes between air-liquid-interfaces (e.g. lung barrier) and liquid-liquid-interfaces (e. g. intestinal barrier). To investigate and evaluate the mechanisms of interaction and transport of NM at biological barriers and their mucus layer, in vitro- and ex vivo-model systems were developed to simulate the exposure of the protective barrier of the organism with environmental relevant concentrations and thus to quantify the induced cellular effects. The focus is currently on environmental relevant, low-dose exposure and long-term studies.
Besides the skin and the gastrointestinal tract, the lung is the most critical organ regarding NM absorption, with a deposition rate of 70 %. The inhalative exposure is of primary importance for potential risk for humans. Within the framework of the project NanoUmwelt, the Fraunhofer IBMT investigates the interactions of NM with the lung barrier and the intestinal barrier. To understand the mechanisms of NM transport over the mentioned barriers in-house developed 2D- and 3D-co-culture models were used, but also primary porcine tissue. One objective is the combination of the cellular models with non-invasive analysis methods to realize real-time multiparameter analysis. To study the transfer of NM over the lung barrier, a co-culture comprising lung and immune cells is cultivated on a porous Polystyrol membrane (Transwell technology). For simulating lung-air interactions, the cells are supplied with nutrients from the basolateral side (bottom side) and treated with NM-containing clouds of artificial aerosol from the apical side (top side).
Using these models nanotoxicological studies in low-dose range are performed, for better assessing the potential risk raising from NM to human health and the environment. IBMT works together with the Fraunhofer Institute for Molecular Biology and Applied Ecology IME, which used pluripotent stem cells to develop a model for investigating cardiotoxicity. Empa, the Swiss partner in the project, delivered a placental barrier model for studying the transport of NM between mother and child.
The NanoUmwelt research project was launched in October 2014 and will last for 36 months. Its objective is to develop methods for detecting minute amounts of NMs in environmental samples. Using this information, the project partners will to assess the effect of NM on humans, animals, and the environment. They are focusing on commercially significant, slowly degradable, metallic (silver, titanium dioxide), carbonic (carbon nanotubes) and polymer-based (polystyrene) NM. The project contributes to the risk assessment, presdiction and evaluation of potential human and ecotoxicity of NM.
The German Federal Ministry for Education and Research (BMBF) is providing the NanoUmwelt project with 1.8 million euros of funding as part of its NanoCare program. Led by Postnova Analytics GmbH, ten further partners are collaborating together on the project. Besides the Fraunhofer Institutes for Biomedical Engineering IBMT and for Molecular Biology and Applied Ecology IME, these partners include Germany’s Environment Agency, Empa (the Swiss Federal Laboratories for Materials Science and Technology), PlasmaChem GmbH, Senova GmbH (biological sciences and engineering), fzmb GmbH (Research Centre of Medical Technology and Biotechnology), the universities of Trier and Frankfurt, and the Rhine Water Control Station in Worms.