Monitoring the composition, sources and spatial distribution of seafloor litter in the Adriatic Sea (Mediterranean Sea) through Fishing for Litter initiatives.

Although the seafloor is an important sink for marine litter, its less accessible environment makes seafloor litter the least studied component. Nevertheless, detailed monitoring of its composition and spatial distribution is urgently needed to develop appropriate mitigation strategies in areas exposed to multiple anthropogenic pressures, such as the Adriatic Sea. Commercial fisheries such as bottom trawls can serve as an opportunistic platform for collecting data on seafloor litter and help researchers in addressing the lack of data through Fishing for Litter initiatives. In this study, the crews of twelve trawlers from two Italian Adriatic ports collected seafloor litter as part of their Fishing for Litter initiatives. In addition to the classical scheme, the collected litter was sorted on board into different bags, and speed, duration and geographical coordinates of each haul were recorded to allow an assessment of both densities and spatial distribution. More than 600 kg of litter was removed from the seafloor by the fishers in approximately 1 month and characterised by the researchers on the docks. Most of the found items were made of synthetic polymers and related to packaging, fisheries and aquaculture activities. Abandoned, lost or discarded fishing gear and mussel nets were identified as of particular concern in the maritime compartment of Chioggia and Civitanova Marche, respectively. Three hotspots for marine litter were identified: near the Venice lagoon, in the centre of the northern basin and at greater depth in the central Adriatic. This study provides a detailed picture of the spatial distribution and composition of seafloor litter in the areas studied and highlights the importance of increased cooperation between fishermen and scientists to improve the identification of hotspots and sources while removing marine litter from the seafloor and raising awareness of the problem.

First Documented Uses of Caves along the Coast of Albania by Mediterranean Monk Seals (Monachus monachus, Hermann 1779): Ecological and Conservation Inferences.

Information on the habitat use of the Mediterranean monk seal (Monachus monachus) along the coast of Albania (Adriatic and Ionian Sea) has so far been limited to vague and generalised data. A survey conducted in the National Marine Park Karaburun-Sazan in the summer of 2019 identified two marine caves with morphological characteristics best suited for use by such species. The two caves were subsequently equipped with infrared camera traps in 2020. The recovery of a scat in one of the caves during the 2019 survey and the photographic material obtained confirmed the use of the cave. This research provides the first documentation of marine cave habitat use by the Mediterranean monk seal in Albania. Quantitative and qualitative assessment of specimens frequenting the area could not be performed due to the limited data obtained on seal presence along the Albanian coasts. Nevertheless, the retrieved information is relevant for Albania and for the species conservation. The collected scat was analysed for trophic and anthropogenic contamination data. Three species (gilthead sea bream, European sea bass, and garfish), as well as four anthropogenic items (including a piece of nylon net), were identified. The inferences resulting from the analyses of the data presented in this study provided additional information on the ecology of the species and its conservation priorities, which need to be contextualized at the Adriatic-Ionian regional scale.

Micro- and mesoplastics in sea surface water from a Northern Adriatic coastal area.

The presence of microplastics in the sea is a global issue widely studied and discussed in the last years. The whole marine ecosystem is now considered at high risk because of their presence and abundance in every studied environment all over the world because polymeric materials commonly constitute the main raw materials in contemporary industrial production. The presented study reports the results obtained from surface seawater monitoring of two sampling transects in the coastal area close to the Venice Lagoon (Italy) inlet, investigated in order to get new information about the presence and relevance of plastic pollution. Plastic particles collected by means of a manta net (0.3-mm mesh size) have been characterized in detail by utilizing a multi-technique approach in order to discriminate them by typology, dimension, colour, spatial density and chemical composition. Such information permitted the individuation of subgroups (specific groups) of plastic micro-debris in this Northern Adriatic area.

Surface Enhanced Raman Spectroscopy With Electrodeposited Copper Ultramicro-Wires With/Without Silver Nanostars Decoration.

The electrochemical preparation of arrays of copper ultramicrowires (CuUWs) by using porous membranes as templates is critically revisited, with the goal of obtaining cheap but efficient substrates for surface enhanced Raman spectroscopy (SERS). The role of the materials used for the electrodeposition is examined, comparing membranes of anodized aluminum oxide (AAO) vs. track-etched polycarbonate (PC) as well as copper vs. glassy carbon (GC) as electrode material. A voltammetric study performed on bare electrodes and potentiostatic tests on membrane coated electrodes allowed the optimization of the deposition parameters. The final arrays of CuUWs were obtained by chemical etching of the template, with NaOH for AAO and CH2Cl2 for PC. After total etching of the template, SERS spectra were recorded on CuUWs using benzenethiol as SERS probe with known spectral features. The CuUW substrates displayed good SERS properties, providing enhancement factor in the 103-104 range. Finally, it was demonstrated that higher Raman enhancement can be achieved when CuUWs are decorated with silver nanostars, supporting the formation of SERS active hot-spots at the bimetallic interface.

Fiber Optic Reflection Spectroscopy-Near-Infrared Characterization Study of Dry Pigments for Pictorial Retouching.

A deep comprehension of composition of pigments, employed nowadays in the field of pictorial retouch is considered essential for a deeper knowledge of their behavior with time once applied on artifacts. A commercially available set consisting of 27 pigments employed for the conservation of both historical and contemporary artworks has been characterized through Reflectance Spectroscopy in the VIS and NIR spectral range. The pigments included in the investigated set are classified into four categories: (i) dyes and colors from plants, (ii) modern pigments, (iii) pigments of own production and historical pigments, and (iv) natural earths. Recorded spectra were interpreted with the aim to detect existing coloring and filling phases and obtained results were compared with available data sheets: some inconsistencies were found, as well as lack of some compounds among the reported ones. Attributions were found for many features, even if in some cases detailed information for a comparison was not found in the literature, especially regarding NIR spectra. The proposed paper aims to provide a useful tool for the study of real artworks with a detailed overview of material characteristics in the visible and near infrared spectral range.

Polyphenolic profile of Cichorium intybus L. endemic varieties from the Veneto region of Italy.

The present study was aimed at the investigation, through HPLCDAD-ESI-MS/MS, of polyphenols in seven autochthonous C. intybus varieties, already known from literature to contain various substances with antioxidant properties, from the Veneto region of Italy, namely 'Castelfranco', 'Chioggia', 'Rosa di Gorizia', 'Rosa di Verona', 'Treviso Precoce', 'Treviso Tardivo' and 'Verdon da Cortèl'. Thirteen polyphenols, belonging to hydroxycinnamic acid, flavone, flavonol and anthocyanin classes, were detected in most samples. The developed analytical method was validated in agreement with ICH guidelines. The total amount of polyphenols ranged from 52 to 386 (mean: 254) mg/100g fresh weight (F.W.). The results were further confirmed by Principal Composition Analysis (PCA), which highlighted peculiar features and similarities among analysed samples for each variety (except for 'Chioggia' samples). The developed method is suitable for routine analyses, as well as geographical characterization, selection of different C. intybus varieties and for the determination of related polyphenols dietary recommended intakes.

Lorenzo Lotto's painting materials: an integrated diagnostic approach.

This paper presents the results of a comprehensive diagnostic investigation carried out on five paintings (three wood panels and two paintings on canvas) by Lorenzo Lotto, one of the most significant artists of the Italian Renaissance in the first half of 16th century. The paintings considered belong to 1508-1522 period, corresponding to the most significant years of Lotto's evolution. A wide array of non-invasive (reflectance spectrometry and X-ray fluorescence) and micro-invasive analytical techniques (optical microscopy, scanning electron microscopy with energy dispersive spectroscopy, micro-FTIR spectroscopy, micro-Raman spectroscopy, gas chromatography coupled with mass spectrometry and high performance liquid chromatography coupled with photodiode array detection and mass spectrometry) were applied in order to provide a large set of significant data, limiting as much as possible the sampling. This study has proved that Lotto's painting palette was typical of Venetian practice of that period, but some significant peculiarities emerged: the use of two kinds of red lakes, the addition of calcium carbonate and colourless powdered glass, the latter frequently found in pictorial and ground layers. Moreover, the integrated investigation showed that Lotto's technique was sometimes characterized by the use of coloured priming and multi-layer sequences with complex mixtures. Chromatographic analyses allowed to identify in all specimens: azelaic, palmitic and stearic acids, generally referring to the presence of drying oils. The extension of additional non-invasive examination to about 50 paintings by the same author, spanning from 1505 to around 1556, helped to verify the evolution in the use of some pigments, such as the yellow ones, where Pb-Sb yellow was used alongside Pb-Sn yellow.

Effects of alginate on stability and ecotoxicity of nano-TiO2 in artificial seawater.

The large-scale use of titanium dioxide nanoparticles (nano-TiO2) in consumer and industrial applications raised environmental health and safety concerns. Potentially impacted ecosystems include estuarine and coastal organisms. Results from ecotoxicological studies with nano-TiO2 dispersed in salt exposure media are difficult to interpret due to fast flocculation and sedimentation phenomena affecting the dispersion stability. The goal of this study was to investigate the stabilisation effect of alginate on uncoated nano-Ti22 in artificial seawater dispersions used in ecotoxicity bioassays. The most effective stabilisation was obtained at alginate concentration of 0.45 g/L after sonicating dispersions for 20 min (100 W). The size distribution remained constant after re-suspension, indicating that no agglomeration occurred after deposition. Ecotoxicity tests on Artemia franciscana and Phaeodactylum tricornutum did not show any adverse effects related to the presence of alginate in the exposure media, and provided evidence on possible reduced bioavailability of nano-TiO2. The suitable concentration of alginate is recommended to occur on a case-by-case basis.

Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model.

A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 µg/cm(2) exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9-13). PLGA-PEO NPs were not toxic up to dose 3 µg/cm(2); dose of 75 µg/cm(2) displays significant decrease in [(3)H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done.

Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles.

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.

Co-exposure to n-TiO2 and Cd2+ results in interactive effects on biomarker responses but not in increased toxicity in the marine bivalve M. galloprovincialis.

The increasing production of nanoparticles (NPs) will lead to their release into the aquatic environment, where they could modify the bioavailability/bioconcentration and consequent biological impact of other contaminants. Interactive effects of n-TiO2, one of the most widespread NP type, and Cd(2+), a common heavy metal pollutant, have been described in freshwater species, whereas no information is available in marine organisms. In this work, the effects of co-exposure to n-TiO2 and Cd(2+) were investigated in the marine bivalve Mytilus galloprovincialis. Experimental conditions (100 µg/L, 96 h), were chosen in order to induce early but measurable stress responses (biomarkers) without toxicity. Several biomarkers, from molecular to tissue level, were measured in hemolymph and digestive gland; the effects on embryo development were also evaluated. In hemolymph, Cd(2+) abolished the increase in immune parameters induced by n-TiO2 (NO production and lysozyme activity). In the digestive gland, distinct interactive effects of n-TiO2 and Cd(2+) were observed on different lysosomal biomarkers (lysosomal membrane stability, lipid accumulation and lysosome/cytoplasm volume ratio) and transcription of the immune genes lysozyme and toll-like receptor (TLR). However, n-TiO2 did not affect specific metal-induced responses (metallothionein induction) and tissue metal accumulation. Cd(2+) alone, but not in combination with n-TiO2, affected embryo development. The interactive effects observed on different biomarkers were not apparently due to differences in bioavailability/bioaccumulation of Cd(2+) in the presence of n-TiO2 agglomerates; these effects may result from interactions of either contaminant with both common and distinct targets/mechanisms of action at different levels of biological organization. Overall, the results indicate that co-exposure to n-TiO2 and Cd(2+) did not result in increased adverse effects in M. galloprovincialis. These data underline the need for further knowledge on the potential interactions of NPs with existing contaminants in marine organisms.

Harmful algae records in Venice lagoon and in Po River Delta (northern Adriatic Sea, Italy).

A detailed review of harmful algal blooms (HAB) in northern Adriatic Sea lagoons (Po River Delta and Venice lagoon) is presented to provide "updated reference conditions" for future research and monitoring activities. In the study areas, the high mollusc production requires the necessity to identify better methods able to prevent risks for human health and socioeconomical interests. So, an integrated approach for the identification and quantification of algal toxins is presented by combining microscopy techniques with Liquid Chromatography coupled with High Resolution Time of Flight Mass Spectrometry (HPLC-HR-TOF-MS). The method efficiency was first tested on some samples from the mentioned coastal areas, where Dinophysis spp. occurred during summer in the sites directly affected by seawaters. Although cell abundance was always <200 cells/L, the presence of Pectenotoxin-2 (PTX2), detected by HPLC-HR-TOF-MS, indicated the potential release of detectable amounts of toxins even at low cell abundance.

Effects of in vitro exposure to titanium dioxide on DNA integrity of bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes.

In the present study, the genotoxic potential of nanosized TiO2 anatase and micro-sized rutile on bottlenose dolphin (Tursiops truncatus) fibroblasts and leukocytes was investigated. Human and mouse cells were also studied in order to compare susceptibility to TiO2 in different mammalian species. Cell lines were exposed for 4, 24, and 48 h to different concentrations of TiO2 (20, 50, 100, 150 µg/ml) and DNA damage was investigated by single cell gel electrophoresis (Comet assay). Both anatase and rutile induced increased DNA damage, even though statistically significant effects were scattered according to species and cell lines. Bottlenose dolphin leukocytes and murine fibroblasts exhibited increased DNA damage after rutile exposure at some doses/times, while human fibroblasts showed a significant dose-response effect after a 4 h exposure to anatase. Human leukocytes were tolerant to both anatase and rutile. Ultrastructural investigation showed that TiO2 particles entered the cell and were compartmentalized within membrane-bound vesicles.

Comprehensive assessment of nephrotoxicity of intravenously administered sodium-oleate-coated ultra-small superparamagnetic iron oxide (USPIO) and titanium dioxide (TiO2) nanoparticles in rats.

As a main excretory organ, kidney is predisposed to direct/indirect injury. We addressed the potential nephrotoxic effects following expositions of healthy rats to nanoparticle (NP) loads relevant to humans in a situation of 100% bioavailability. Up to 4 weeks after administration, a single iv bolus of oleate-coated ultra-small superparamagnetic iron oxide NPs (in dose of 0.1%, 1.0% and 10.0% of LD50) or TiO2 NPs (1.0% of LD50) did not elicit decline in renal function, damage to proximal tubules, alterations in: renal histology or expression of pro-inflammatory/pro-fibrotic genes, markers of systemic or local renal micro-inflammation or oxidative damage. Antioxidant enzyme activities in renal cortex, mildly elevated at 24 h, completely restored at later time points. Data obtained by multifaceted approach enable the prediction of human nephrotoxicity during preclinical studies, and may serve as comparison for alternative testing strategies using in vitro and in silico methods essential for the NP-nephrotoxicity risk assessment.

Determination of benzotrifluoride derivative compounds in groundwater.

Two simple analytical methods for the simultaneous determination and quantification of benzotrifluoride and eight chlorinated, amino and nitro benzotrifluoride derivatives in groundwater are proposed. Benzotrifluoride, 4-chlorobenzotrifluoride, 2,4-dichlorobenzotrifluoride and 3,4-dichlorobenzotrifluoride, were extracted by Purge-and-Trap on the basis of their volatile properties, while 3-aminobenzotrifluoride, 4-nitrobenzotrifluoride, 3-amino-4-chlorobenzotrifluoride, 3-nitro-4-chlorobenzotrifluoride and 4-chloro-3,5-dinitrobenzotrifluoride extractions were done with an automated SPE system. The analytical separations and detections were performed with two different GC systems, both equipped with single quadrupole mass spectrometer as detector. The LOD ranges for the two methods were 0.002-0.005 µg L(-1) and 0.01-0.07 µg L(-1), respectively. Both extraction methods were developed using spiked Milli-Q water and were then demonstrated with groundwater samples collected during autumn 2008. The areas of groundwater collection were polluted due to an episode of improper industrial soil disposal and consequent leakage of aliphatic and aromatic, fluorinated chemicals into the groundwater. This work eventually revealed the presence of several benzotrifluoride compounds most of them, like dichloro- and amino-derivatives, never been reported as environmental contaminants.

Effects of lung exposure to carbon nanotubes on female fertility and pregnancy. A study in mice.

We studied the effects of preconceptional exposure to multiwalled carbon nanotubes (MWCNTs): mature, female C57BL/6J mice were intratracheally instilled with 67µg NM-400 MWCNT, and the following day co-housed with mature males, in breeding pairs. Time to delivery of the first litter, litter parameters, maternal inflammation and histopathology of lung and liver were recorded. In male offspring, locomotor activity, startle response, and daily sperm production (DSP) were assessed. In the dams, lung and liver bore evidence of MWCNT exposure when assessed 6 weeks and 4 months after exposure. A short delay in the delivery of the first litter was observed in exposed females. Litter parameters, behavior and DSP were similar in control and exposed groups. In conclusion, instillation of a single dose of MWCNT induced long lasting pathological changes in dam lung and liver. Theoretically, lung inflammation due to particle exposure could interfere with female reproductive parameters. Whether the observed lag in delivery of a first litter was in fact caused by exposure to MWCNT should be addressed in a study designed specifically to elucidate effects on the early processes involved in establishment of pregnancy. Exposure was not associated with changes in the assessed gestational or offspring parameters.

In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve Mytilus galloprovincialis.

Due to the increasing production of nanoparticles (NPs) and their potential release in the aquatic environment, evaluation of their biological impact on aquatic organisms represents a major concern. Suspension feeding invertebrates, in particular bivalve mollusks, may play a role in NP biotransformation and transfer through food webs and may represent a significant target for NP toxicity. In this work, the in vivo effects of titanium dioxide (n-TiO2), one of the most widespread NPs in use, were investigated in the bivalve Mytilus galloprovincialis, largely utilised as a sentinel for marine contamination. Mussels were exposed for 96h to different concentrations of n-TiO2 suspensions (1, 10 and 100µgL(-1)) and multiple responses were evaluated in the digestive gland and immune cells, the haemocytes. In the digestive gland, n-TiO2 affected lysosomal and oxidative stress biomarkers and decreased transcription of antioxidant and immune-related genes. In the haemocytes, n-TiO2 decreased lysosomal membrane stability-LMS and phagocytosis, increased oxyradical production and transcription of antimicrobial peptides; moreover, pre-apoptotic processes were observed. The effects of n-TiO2 on digestive gland and haemocytes were distinct, also depending on the endpoint and on nominal NP concentrations, with many significant responses elicited by the lowest concentrations tested. The results show that n-TiO2, at concentrations close to predicted environmental levels, significantly affected different functional and molecular parameters of mussel digestive gland and immune cells. In particular, the observed changes in immune parameters that represent significant biomarkers of exposure at the organism level suggest that exposure to n-TiO2 may pose a serious risk to mussel health.

In vitro assessment of engineered nanomaterials using a hepatocyte cell line: cytotoxicity, pro-inflammatory cytokines and functional markers.

Effects on the liver C3A cell line treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO; coated 100 nm and uncoated 130 nm), two multi-walled carbon nanotubes (MWCNTs), one silver (Ag < 20 nm), one 7 nm anatase, two rutile TiO2 nanoparticles (10 and 94 nm) and two derivatives with positive and negative covalent functionalisation of the 10 nm rutile were evaluated. The silver particles elicited the greatest level of cytotoxicity (24 h LC50 - 2 µg/cm(2)). The silver was followed by the uncoated ZnO (24 h LC50 - 7.5 µg/cm(2)) and coated ZnO (24 h LC50 - 15 µg/cm(2)) particles with respect to cytotoxicity. The ZnO NMs were found to be about 50-60% soluble which could account for their toxicity. By contrast, the Ag was <1% soluble. The LC50 was not attained in the presence of any of the other engineered NMs (up to 80 µg/cm(2)). All NMs significantly increased IL-8 production. Meanwhile, no significant change in TNF-α, IL-6 or CRP was detected. Urea and albumin production were measured as indicators of hepatic function. These markers were only altered by the coated and uncoated ZnO, which significantly decreased albumin production.

Engineered nanomaterial risk. Lessons learnt from completed nanotoxicology studies: potential solutions to current and future challenges.

PARTICLE_RISK was one of the first multidisciplinary projects funded by the European Commission's Framework Programme that was responsible for evaluating the implications of nanomaterial (NM) exposure on human health. This project was the basis for this review which identifies the challenges that exist within the assessment of NM risk. We have retrospectively reflected on the findings of completed nanotoxicology studies to consider what progress and advances have been made within the risk assessment of NMs, as well as discussing the direction that nanotoxicology research is taking and identifying the limitations and failings of existing research. We have reflected on what commonly encountered challenges exist and explored how these issues may be resolved. In particular, the following is discussed (i) NM selection (ii) NM physico-chemical characterisation; (iii) NM dispersion; (iv) selection of relevant doses and concentrations; (v) identification of relevant models, target sites and endpoints; (vi) development of alternatives to animal testing; and (vii) NM risk assessment. These knowledge gaps are relatively well recognised by the scientific community and recommendations as to how they may be overcome in the future are provided. It is hoped that this will help develop better defined hypothesis driven research in the future that will enable comprehensive risk assessments to be conducted for NMs. Importantly, the nanotoxicology community has responded and adapted to advances in knowledge over recent years to improve the approaches used to assess NM hazard, exposure and risk. It is vital to learn from existing information provided by ongoing or completed studies to avoid unnecessary duplication of effort, and to offer guidance on aspects of the experimental design that should be carefully considered prior to the start of a new study.