Principle and scope. The original SOP “Final protocol for producing suitable manufactured nanomaterial exposure media” was developed as a generic approach for the preparation of batch dispersions for in vitro and in vivo toxicity testing in the NANOGENOTOX project [21]. The method aims to produce a highly dispersed state of any MN by ethanol (EtOH) pre-wetting to handle hydrophobic materials followed by dispersion in sterile-filtered 0.05% w/v BSA-water at a fixed concentration of 2.56 mg/ml using probe sonication. The protocol may not produce the smallest possible particle size in the dispersion, but is a generically applicable procedure that ensures reasonable dispersion of the test materials selected for the NANOGENOTOX project, with the aim to use or characterize the dispersion immediately after its production [21]. To precisely and accurately measure the size and shape of (nano)materials in the context of the EC definition [22] using TEM, it is efficient to modify the generic dispersion protocol. For the examined materials and conditions, a minor modification omitting the pre-wetting step with EtOH and the treatment with 0.05% w/v BSA-kept the particles in a highly dispersed state, allowed representative transfer to an EM-grid, and it improved the separation of the particles from the background based on mass-density contrast and the identification of the primary particles in aggregates.
Principle and scope. The proposed method for preparation of EM specimens brings particles in dispersion in contact with an EM-grid and allows them to interact with the grid surface. When excess fluid is drained of and grids are air-dried, a fraction of the particles remains attached to the grid by different types of interactions (electrostatic, apolar, van der Waals). This procedure aims to prepare a TEM specimen from dispersed particles. The concentrations of particles, and the type and charge of the grid are chosen such that the fraction of nanoparticles attached to the grids optimally represents the dispersed particles, and that the particles of interest can be detected individually. Transfer of the particles to the grid is not complete such that absolute counts cannot be realized. The SOP is useful for particles that can be metallic, metal oxides or other. The particles can be monodisperse or polydisperse, aggregated or not. The medium can be polar (water, phosphate buffered saline,...) or apolar (hexane, acetone,…). The prepared EM specimens are useful for descriptive TEM analyses or quantitative TEM analyses. To be suitable for quantitative TEM analysis, the particles should be evenly distributed over the grids, while the fraction of the attached NPs represents the dispersed particles optimally.
Principle and scope. The proposed method for TEM imaging of nanomaterials aims to record a set of calibrated transmission electron micrographs showing particles that are representative for the NM on the EM grid starting from EM specimens containing particles that optimally represent the particles in the original sample, and that contain particles of interest that can be detected individually. The TEM specimen preparation can be performed based on the grid-on-drop or drop-on-grid methods described in 3.2 , or on other specimen preparation methods including cryo-EM, aerosol sampling and on grid ultracentrifugation centrifugation. To assure unbiased random image collection the systematic micrograph selection procedure of Xx Xxxxxxxxx et al. [11] is applied. The magnification of the micrographs and the number of particles (micrographs) are determined such that the images are suitable for subsequent descriptive and quantitative image analyses. The pixel size and the associated magnification is determined based on the criterion of Merkus [53]. The upper size detection limit is limited to one tenth of the image size supporting on ISO 13322-1, 2014 [54]. The number of particles required to estimate a quantitative parameter with a certain confidence level is determined based on the method proposed by Xx Xxxxxxxxx et al. [9, 51].
Principle and scope. A descriptive or qualitative EM analysis aims to provide a description of specific physical properties of a nanomaterial that determine, among others, its interaction with biological and environmental systems based on calibrated, bright-field TEM or SEM micrographs taken at low to intermediate magnification. It allows evaluating under which physical form the NM is exposed to in vivo and in vitro test systems, and whether a subsequent quantitative TEM analysis of the NM is feasible. The procedure is based on guidelines described in literature [70, 78-82]. In principle this method allows to describe the characteristics of any kind of nanoparticles. A descriptive EM analysis includes (i) an estimate of the size (distribution) of the primary and aggregated/agglomerated particles: (ii) representative and calibrated micrographs; (iii) the agglomeration- and aggregation status; (iv) the general morphology; (v) the surface topology; (vi) the structure (crystalline, amorphous, …); (vii) and the presence of contaminants and aberrant particles. In addition, such qualitative analysis evaluates the relevance and suitability of a quantitative TEM analysis based on the amount of particles on the EM grid and the homogeneity of their distribution. The proposed methodology complies with the EFSA Guidance document that foresees application of electron microscopy (TEM) such that the generated data is in line with the current Guidance document. It describes several key parameters important to assess the nanoparticle safety as specified in [28, 29, 63, 64].
Principle and scope. This procedure aims to analyse the 2D properties of the particles on EM micrographs. To be suitable for quantitative characterisation, the images should have a homogeneous background and the particles should be clearly distinguishable from the background. This method allows characterising NM on EM-micrographs using image analysis software. The NM can be metallic consisting for example of Ag or Au, an oxide including SiO2, TiO2,Fe2O3, Fe3O4 ,and other. The NM can be monodisperse or polydisperse. Freeware and/or commercial image analysis softwares can be applied.
Principle and scope. This procedure aims to analyse the 2D properties of the primary particles on EM micrographs. The image analysis program detects aggregated particles on an EM micrograph based on their grey value, which reflects the mass-density contrast of the material. Aggregates that are distinguishable from the background are detected and semi-automatically measured. Multiple measurands are measured simultaneously on individual aggregates. The primary particles in the aggregates are detected based on watershed segmentation and their minimal size and overlap coefficient are measured based on an Euclidean distance map. A typical particle analysis consists of following steps: Image preparation Setting and adjusting the threshold value Defining the detection area Setting the detection parameters Detection of the primary particles in aggregated NM Selection of the primary particle parameters Defining the classification schemes Classification of the particles according the selected parameters Exporting of results in excel spreadsheets and storage of the (annotated) images in the NM database
Principle and scope. 1. Member States shall include in their CAP Strategic Plans a system of conditionality, under which farmers and other beneficiaries receiving direct payments under Chapter II of this Title or the annual payments under Articles 65, 66 and 67 shall be subject to an administrative penalty if they do not comply with the statutory management requirements under Union law and the GAEC standards established in the CAP Strategic Plan, as listed in Annex III, relating to the following specific areas:
Principle and scope. 1. At the latest by 1/1/2025, Member States shall include in their CAP Strategic Plans that farmers and other beneficiaries receiving direct payments under Chapter II of this Title or the annual payments under Articles 65, 66 and 67 of this Regulation shall be subject to an administrative penalty if they do not comply with the requirements related to applicable working and employment conditions or employer obligations arising from the legal acts referred to in Annex XX.
