Martin Hassellöv

Detection and characterization of engineered nanoparticles in food and the environment.

Nanotechnology is developing rapidly and, in the future, it is expected that increasingly more products will contain some sort of nanomaterial. However, to date, little is known about the occurrence, fate and toxicity of nanoparticles. The limitations in our knowledge are partly due to the lack of methodology for the detection and characterisation of engineered nanoparticles in complex matrices, i.e. water, soil or food. This review provides an overview of the characteristics of nanoparticles that could affect their behaviour and toxicity, as well as techniques available for their determination. Important properties include size, shape, surface properties, aggregation state, solubility, structure and chemical composition. Methods have been developed for natural or engineered nanomaterials in simple matrices, which could be optimized to provide the necessary information, including microscopy, chromatography, spectroscopy, centrifugation, as well as filtration and related techniques. A combination of these is often required. A number of challenges will arise when analysing environmental and food materials, including extraction challenges, the presence of analytical artifacts caused by sample preparation, problems of distinction between natural and engineered nanoparticles and lack of reference materials. Future work should focus on addressing these challenges.

Nanoparticle analysis and characterization methodologies in environmental risk assessment of engineered nanoparticles

Environmental risk assessments of engineered nanoparticles require thorough characterization of nanoparticles and their aggregates. Furthermore, quantitative analytical methods are required to determine environmental concentrations and enable both effect and exposure assessments. Many methods still need optimization and development, especially for new types of nanoparticles in water, but extensive experience can be gained from the fields of environmental chemistry of natural nanomaterials and from fundamental colloid chemistry. This review briefly describes most methods that are being exploited in nanoecotoxicology for analysis and characterization of nanomaterials. Methodological aspects are discussed in relation to the fields of nanometrology, particle size analysis and analytical chemistry. Differences in both the type of size measures (length, radius, aspect ratio, etc.), and the type of average or distributions afforded by the specific measures are compared. The strengths of single particle methods, such as electron microscopy and atomic force microscopy, with respect to imaging, shape determinations and application to particle process studies are discussed, together with their limitations in terms of counting statistics and sample preparation. Methods based on the measurement of particle populations are discussed in terms of their quantitative analyses, but the necessity of knowing their limitations in size range and concentration range is also considered. The advantage of combining complementary methods is highlighted.

Considerations for environmental fate and ecotoxicity testing to support environmental risk assessments for engineered nanoparticles

There is an increasing concern over the safety of engineered nanoparticles (ENPs) to humans and the environment and it is likely that the environmental risks of these particles will have to be tested under regulatory schemes such as REACH. Due to their unique properties and the fact that their detection and characterisation in complex matrices is challenging, existing analytical methods and test approaches for assessing environmental risk may not be appropriate for ENPs. In this article we discuss the challenges associated with the testing of ENPs to generate data on persistence, mobility, bioavailability and ecotoxicity in the environment. It is essential that careful consideration is given to the selection of the test material, the test system (including test vessels and study media) and the test exposure conditions. During a study it is critical that not only the concentration of the ENP is determined but also its characteristics (e.g. size, shape, degree of aggregation and dissolution). A range of analytical techniques is available including microscopy-based approaches (e.g transmission and scanning electron microscopy), dynamic light scattering, and size separation approaches (e.g. field flow fractionation and hydrodynamic chromatography) coupled to detection methods such as inductively coupled plasma MS. All of these have their disadvantages: some are unable to distinguish between ENPs and natural interferences; some techniques require sample preparation approaches that can introduce artefacts; and others are complex and time-consuming. A combination of techniques is therefore needed. Our knowledge in this area is still limited, and co-ordinated research is required to gain a better understanding of the factors and processes affecting ENP fate and effects in the environment as well as to develop more usable, robust and sensitive methods for characterisation and detection of ENPs in environmental systems.

Nanomaterials for environmental studies: Classification, reference material issues, and strategies for physico-chemical characterisation

NanoImpactNet is a European Commission Framework Programme 7 (FP7) funded project that provides a forum for the discussion of current opinions on nanomaterials in relation to human and environmental issues. In September 2008, in Zurich, a NanoImpactNet environmental workshop focused on three key questions: 1. What properties should be characterised for nanomaterials used in environmental and ecotoxicology studies? 2. What reference materials should be developed for use in environmental and ecotoxicological studies? 3. Is it possible to group different nanomaterials into categories for consideration in environmental studies? Such questions have been, at least partially, addressed by other projects/workshops especially in relation to human health effects. Such projects provide a useful basis on which this workshop was based, but in this particular case these questions were reformulated in order to focus specifically on environmental studies. The workshop participants, through a series of discussion and reflection sessions, generated the conclusions listed below. The physicochemical characterisation information identified as important for environmental studies included measures of aggregation/agglomeration/dispersability, size, dissolution (solubility), surface area, surface charge, surface chemistry/composition, with the assumption that chemical composition would already be known. There is a need to have test materials for ecotoxicology, and several substances are potentially useful, including TiO(2) nanoparticles, polystyrene beads labelled with fluorescent dyes, and silver nanoparticles. Some of these test materials could then be developed into certified reference materials over time. No clear consensus was reached regarding the classification of nanomaterials into categories to aid environmental studies, except that a chemistry-based classification system was a reasonable starting point, with some modifications. It was suggested, that additional work may be required to derive criteria that can be used to generate such categories, that would also include aspects of the material structure and physical behaviour.

Characterization of the effluent from a nanosilver producing washing machine

The increasing number of nanomaterial based consumer products raises concerns about their possible impact on the environment. This study provides an assessment of the effluent from a commercially available silver nanowashing machine. The washing machine released silver in its effluent at an average concentration of 11μgL(-1), as determined by inductive coupled mass spectrometry (ICP-MS). The presence of silver nanoparticles (AgNPs) was confirmed by single particle ICP-MS as well as ion selective electrode measurements and filtration techniques. Size measurements showed particles to be in the defined nanosize range, with an average size of 10nm measured with transmission electron microscopy (TEM) and 60-100nm determined with nanoparticle tracking analysis (NTA). The effluent was shown to have negative effects on a natural bacterial community as its abundance was clearly reduced when exposed to the nanowash water. If washing machines capable of producing AgNPs become a common feature of households in the future, wastewater will contain significant loadings of AgNPs which might be released into the environment.

Competition between iron- and carbon-based colloidal carriers for trace metals in a freshwater assessed using flow field-flow fractionation coupled to ICPMS

Abstract Flow field–flow fractionation (FlFFF) coupled to an inductively coupled plasma mass spectrometer (ICPMS) has been used to determine the chemical composition of colloids from a freshwater sample as a function of size. Organic carbon and iron are the most abundant colloidal components, and are considered as the major carrier phases for other chemical elements present. The size distribution of organic carbon colloids shows a single peak with an estimated hydrodynamic diameter between 1 and 1.5 nm, while the iron colloids show a more complex distribution centred at larger colloid sizes with estimated hydrodynamic diameters up to 5 nm. The association of 32 trace elements with these two carrier colloids has been quantified by deconvolution analysis, and the resulting distributions are shown to be chemically consistent. The observed distributions are also shown to be broadly consistent with predictions from speciation modelling for the subset of 8 elements for which appropriate stability constants are available.

Size Discrimination and Detection Capabilities of Single-Particle ICPMS for Environmental Analysis of Silver Nanoparticles

The detection capabilities of single particle inductively coupled plasma-mass spectrometry (spICPMS) with respect to particle size and number concentrations are investigated for the case of silver nanoparticles (ca. 20-80 nm). An iterative algorithm was developed where particle measurement events were distinguished as outliers from the more continuous dissolved ion signal if the measured intensity was more than five times the standard deviation of the whole data set. The optimal dwell time for 40-80 nm particles, limiting both incomplete and multiple particle events, was 5 ms. The smallest detectable particle size (ca. 20 nm) is mainly limited by the overlap of particle events and dissolved signal that increases with noise on both signals. The lowest measurable number concentration is limited by the relative frequency of erroneously identified particle events, a limit that can be reduced by acquiring more data points. Finally, the potential of spICPMS for environmental detection of nanoparticles is demonstrated for a wastewater treatment plant effluent sample.

Rapid, high-precision potentiometric titration of alkalinity in ocean and sediment pore waters

Abstract A system for rapid, high precision potentiometric determination of alkalinity in sea water and sediment pore water is presented. Two titration units were used: a 40 ml unit for seawater and a small volume unit for sediment pore water. Titration time was normally less than 10 minutes per sample, including sample exchange. With a 40 ml sample volume, the relative standard deviation of the alkalinity obtained in the laboratory was 0.05% and at sea 0.1 %. The small-volume system (0.5–1.5 ml) gave a precision of 0.07%. Five titration points, in two groups after the second equivalence point, were used to evaluate the equivalence volume. Results from equilibrium calculations and computer simulated alkalinity titrations show that it was possible to use a non-modified Gran function [( V 0 +v) ∗ 10 ( E Z ) ] and still achieve good accuracy and precision.

Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes.

The use of nanomaterials is rapidly increasing, while little is known about their possible ecotoxicological effects. This work investigates the toxic effects of silver (Ag) and gold (Au) nanoparticles on rainbow trout hepatocytes. In addition to toxicity assessment the particles were characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Hepatocyte primary cultures were exposed to Au and Ag nanoparticles, with and without dissolved organic carbon (DOC), as well as HAuCl(4) and AgNO(3) as ionic solutions at concentrations up to 17.4mg/L and 19mg/L, respectively. Ag and Au particles were within the small nanometer size range when dispersed in pure water. In media with higher ionic strength and DOC, particles tended to agglomerate. Cytotoxicity assessments showed that Ag nanoparticles caused a significant reduction in membrane integrity and cellular metabolic activity in a concentration-dependent manner. Au nanoparticles caused a threefold elevation of ROS levels, but no cytotoxicity occurred at concentrations tested. The addition of DOC did not alter the particles potency of cytotoxicity or ROS induction capacity. The current study shows that Ag and Au nanoparticles have adverse effects on rainbow trout hepatocytes at low mg/L concentrations.

Uptake and effects of manufactured silver nanoparticles in rainbow trout (Oncorhynchus mykiss) gill cells.

Nanoparticles are already widely used in technology, medicine and consumer products, but there are limited data on their effects on the aquatic environment. In this study the uptake and effect of citrate (AgNP(CIT)) and polyvinylpyrrolidone (AgNP(PVP)) coated manufactured silver nanoparticles, as well as AgNO(3) (Ag(+)) were tested using primary gill cells of rainbow trout (Oncorhynchus mykiss). Prior to use, the nanoparticles were characterized for size, surface charge and aggregation behavior. Gill cells were cultured either as monolayers on solid support, or as multilayers on a permeable support cell culturing system, enabling transport studies. The uptake of silver nanoparticles and Ag(+) after exposure to 10 mg L(-1) was determined with microscopical methods and inductively coupled plasma mass spectrometry (ICP-MS). Cytotoxicity, in terms of membrane integrity, as well as oxidative stress (depletion of reduced glutathione) was tested at silver concentrations ranging from 0.1 mg L(-1) to 10 mg L(-1). Results show that AgNP(CIT) nanoparticles are readily taken up into gill cell monolayers while uptake was less for AgNP(PVP). In contrast, it appears that the slightly smaller AgNP(PVP) were transported through cultured multilayers to a higher extent, with transport rates generally being in the ng cm(-2) range for 48 h exposures. Transport rates for all exposures were dependent on the epithelial tightness. Moderate cytotoxic effects were seen for all silver treatments. Levels of reduced glutathione were elevated in contrast to control groups, pointing on a possible overcompensation reaction. Taken together silver nanoparticles were taken up into cells and did cause silver transport over cultured epithelial layers with uptake and transport rates being different for the two nanoparticle species. All silver treatments had measurable effects on cell viability.