Nanotoxicology: 2D analysis in a 3D matrix

Engineered nanoparticles are the fastest growing nanotechnology product, and of increasing environmental concern. Examples include antimicrobial silver nanoparticles, iron oxide particles used in bulk scale for environmental remediation, and micro- and nano-plastics. The latter are added to a broad range of cosmetic products. Larger plastic pieces pollute the environment by breaking down naturally to form smaller particles. These particles may attract and concentrate hydrophobic persistent organic pollutants on their surface, which might later be released once inside a biological matrix, as has been shown indirectly in e.g. birds [1]. All these particles are found in nature and humans are exposed to them daily (e.g. in beer [2]). People who regularly consume cultured bivalves may ingest up to 11.000 micro plastic particles per year by this route alone, even ignoring particles smaller than 5 µm [3]. Classically, nanoparticle uptake studies have been performed by acid digestion and chemical analysis [4]. But how do these particles interact with organisms and their tissue? Are they taken up in the tissue [5], or are they passively passing through the digestion system? Such questions are interesting in particular with regard to cytotoxic particles, such as silver, or very small particles that might enter the nucleus and interfere with DNA replication. Particles with the capability to carry toxic compounds, such as plastic, also have the potential for causing disruption of normal cellular function. In order to further our understanding of how such particles interact with tissue at the cellular level, nanoscale analysis is required, either to verify the nature of the particle (STEM EDX point analysis of single particles, Figure 1 a-c) or to trace substances released from their surface (NanoSIMS). However, before such analysis, a first challenge is to locate the particles in the affected cells. This has proven a major obstacle because of the relatively large size of the organism investigated. Indeed, screening a large volume of the organism under investigation to locate nanoparticles before EDX and NanoSIMS analysis is thus often a large part of the work. We work with model organisms such as microalgae, daphnia and earthworms that are likely to come into contact with, and accumulate nanoparticles. During the past few years we have employed various methods to localize different particles inside the organisms, e.g. serial block-face imaging, FIB-SEM imaging, freeze-dried cryo-sections and serial sections (Figure 1 d). Correlation between screening- and analysis techniques is key to locate, and be able to investigate the effects of nanoparticles in a biological matrix. At the moment we are exploring methods to screen large amounts of serial sections made by a custom-made knife, and methods for transferring interesting sections to carriers compatible with either STEM EDX or NanoSIMS. References [1] K. Tanaka et al., Marine Poll Bull 69 (1-2) (2013) 219. [2] G. Liebezeit and E. Liebezeit, Food Addit. Contam. Part a Chem. Anal. Control Exposure Risk Assess (2014). [3] L. van Cauwenberghe, and C. R. Janssen. Environ Pollut 193 (2014) 65. [4] L. M. Skjolding et al., Ecotoxicology 23 (2014) 1172. [5] P. Rosenkrantz et al., Environ Toxicol Chem 28 (2009) 2142. [6] We would like to thank Irina Kolotueva for invaluable technical advice. This research was supported by The Society of Electron Microscope Technology, and the European Research Council (Grant no. 281579). FIG. 1. (a-c) HAADF STEM images of D. magna gut epithelia exposed to 10 nm gold nanoparticles (Au NP) (0.4 mg Au/L) for 24h. Corresponding EDX spectra are superimposed. For clarity the C-peaks are capped and only up to 2.5 keV is depicted. (a) Au NP at microvilli, scale bar = 100 nm, (b) Au NP at base of microvilli, scale bar = 100 nm, (c) Os-rich particles in gut cell lipid droplet, scale bar = 50 nm, (d) Serial sections of L. variegatus exposed to iron particles on Si-wafer

The Danish contribution to the European DEMOCOPHES project: A description of cadmium, cotinine and mercury levels in Danish mother-child pairs and the perspectives of supplementary sampling and measurements

Human biomonitoring (HBM) is an important tool, increasingly used for measuring true levels of the body burdens of environmental chemicals in the general population. In Europe, a harmonized HBM program was needed to open the possibility to compare levels across borders. To explore the prospect of a harmonized European HBM project, DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) was completed in 17 European countries. The basic measurements performed in all implemented countries of DEMOCOPHES included cadmium, cotinine and phthalate metabolites in urine and mercury in hair. In the Danish participants, significant correlations between mothers and children for mercury in hair and cotinine in urine were found. Mercury in hair was further significantly associated with intake of fish and area of residence. Cadmium was positively associated with BMI in mothers and an association between cadmium and cotinine was also found. As expected high cotinine levels were found in smoking mothers. For both mercury and cadmium significantly higher concentrations were found in the mothers compared to their children. In Denmark, the DEMOCOPHES project was co-financed by the Danish ministries of health, environment and food safety. The co-financing ministries agreed to finance a number of supplementary measurements of substances of current toxicological, public and regulatory interest. This also included blood sampling from the participants. The collected urine and blood samples were analyzed for a range of other persistent and non-persistent environmental chemicals as well as two biomarkers of effect. The variety of supplementary measurements gives the researchers further information on the exposure status of the participants and creates a basis for valuable knowledge on the pattern of exposure to various chemicals.publisher: Elsevier articletitle: The Danish contribution to the European DEMOCOPHES project: A description of cadmium, cotinine and mercury levels in Danish mother-child pairs and the perspectives of supplementary sampling and measurements journaltitle: Environmental Research articlelink: http://dx.doi.org/10.1016/j.envres.2014.07.028 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

Policy recommendations and cost implications for a more sustainable framework for European human biomonitoring surveys

The potential of Human Biomonitoring (HBM) in exposure characterisation and risk assessment is well established in the scientific HBM community and regulatory arena by many publications. The European Environment and Health Strategy as well as the Environment and Health Action Plan 2004–2010 of the European Commission recognised the value of HBM and the relevance and importance of coordination of HBM programmes in Europe. Based on existing and planned HBM projects and programmes of work and capabilities in Europe the Seventh Framework Programme (FP 7) funded COPHES (COnsortium to Perform Human Biomonitoring on a European Scale) to advance and improve comparability of HBM data across Europe. The pilot study protocol was tested in 17 European countries in the DEMOCOPHES feasibility study (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) cofunded (50%) under the LIFE+ programme of the European Commission. The potential of HBM in supporting and evaluating policy making (including e.g. REACH) and in awareness raising on environmental health, should significantly advance the process towards a fully operational, continuous, sustainable and scientifically based EU HBM programme. From a number of stakeholder activities during the past 10 years and the national engagement, a framework for sustainable HBM structure in Europe is recommended involving national institutions within environment, health and food as well as European institutions such as ECHA, EEA, and EFSA. An economic frame with shared cost implications for national and European institutions is suggested benefiting from the capacity building set up by COPHES/DEMOCOPHES.JRC.I.1-Chemical Assessment and Testin

Policy recommendations and cost implications for a more sustainable framework for European human biomonitoring surveys

© 2014 Elsevier Inc. All rights reserved.The potential of Human Biomonitoring (HBM) in exposure characterisation and risk assessment is well established in the scientific HBM community and regulatory arena by many publications. The European Environment and Health Strategy as well as the Environment and Health Action Plan 2004-2010 of the European Commission recognised the value of HBM and the relevance and importance of coordination of HBM programmes in Europe. Based on existing and planned HBM projects and programmes of work and capabilities in Europe the Seventh Framework Programme (FP 7) funded COPHES (COnsortium to Perform Human Biomonitoring on a European Scale) to advance and improve comparability of HBM data across Europe. The pilot study protocol was tested in 17 European countries in the DEMOCOPHES feasibility study (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) cofunded (50%) under the LIFE+ programme of the European Commission. The potential of HBM in supporting and evaluating policy making (including e.g. REACH) and in awareness raising on environmental health, should significantly advance the process towards a fully operational, continuous, sustainable and scientifically based EU HBM programme. From a number of stakeholder activities during the past 10 years and the national engagement, a framework for sustainable HBM structure in Europe is recommended involving national institutions within environment, health and food as well as European institutions such as ECHA, EEA, and EFSA. An economic frame with shared cost implications for national and European institutions is suggested benefitting from the capacity building set up by COPHES/DEMOCOPHES.COPHES was coordinated by BiPRO GmbH, Germany, with the University of Leuven, Belgium and was funded by DG Research in the Seventh Framework Programme (FP7/2007-2013). DEMOCOPHES (LIFE09 ENV/BE/000410) was coordinated by the Federal Public Service Health, Food Chain Safety and Environment, Belgium and was jointly financed by the European Commission LIFE┼ programme (50%) and national institutions in each participating country

Policy recommendations and cost implications for a more sustainable framework for European human biomonitoring surveys

The potential of Human Biomonitoring (HBM) in exposure characterisation and risk assessment is well established in the scientific HBM community and regulatory arena by many publications. The European Environment and Health Strategy as well as the Environment and Health Action Plan 2004–2010 of the European Commission recognised the value of HBM and the relevance and importance of coordination of HBM programmes in Europe. Based on existing and planned HBM projects and programmes of work and capabilities in Europe the Seventh Framework Programme (FP 7) funded COPHES (COnsortium to Perform Human Biomonitoring on a European Scale) to advance and improve comparability of HBM data across Europe. The pilot study protocol was tested in 17 European countries in the DEMOCOPHES feasibility study (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) cofunded (50%) under the LIFE+ programme of the European Commission. The potential of HBM in supporting and evaluating policy making (including e.g. REACH) and in awareness raising on environmental health, should significantly advance the process towards a fully operational, continuous, sustainable and scientifically based EU HBM programme. From a number of stakeholder activities during the past 10 years and the national engagement, a framework for sustainable HBM structure in Europe is recommended involving national institutions within environment, health and food as well as European institutions such as ECHA, EEA, and EFSA. An economic frame with shared cost implications for national and European institutions is suggested benefitting from the capacity building set up by COPHES/DEMOCOPHESAplinkotyros katedraVytauto Didžiojo universiteta