Applicability of OECD TG 201, 202, 203 for the aquatic toxicity testing and assessment of 2D Graphene material nanoforms to meet regulatory needs.

Tests using algae and/or cyanobacteria, invertebrates (crustaceans) and fish form the basic elements of an ecotoxicological assessment in a number of regulations, in particular for classification of a substance as hazardous or not to the aquatic environment according to the Globally Harmonised System of Classification and Labelling of Chemicals (GHS-CLP) (GHS, 2022) and the REACH regulation (Registration, Evaluation, Authorisation and Restriction of Chemicals, EC, 2006). Standardised test guidelines (TGs) of the Organisation for Economic Co-operation and Development (OECD) are available to address the regulatory relevant endpoints of growth inhibition in algae and cyanobacteria (TG 201), acute toxicity to invertebrates (TG 202), and acute toxicity in fish (TG 203). Applying these existing OECD TGs for testing two dimensional (2D) graphene nanoforms may require more attention, additional considerations and/or adaptations of the protocols, because graphene materials are often problematic to test due to their unique attributes. In this review a critical analysis of all existing studies and approaches to testing used has been performed in order to comment on the current state of the science on testing and the overall ecotoxicity of 2D graphene materials. Focusing on the specific tests and available guidance's, a complete evaluation of aquatic toxicity testing for hazard classification of 2D graphene materials, as well as the use of alternative tests in an integrated approach to testing and assessment, has been made. This information is essential to ensure future assessments generate meaningful data that will fulfil regulatory requirements for the safe use of this "wonder" material.

Sphagnum palustre clone vs native Pseudoscleropodium purum: A first trial in the field to validate the future of the moss bag technique.

Although a large body of literature exists on the use of transplanted mosses for biomonitoring of air pollution, no article has addressed so far the use and the accumulation performance of a cloned moss for this purpose. In this work, a direct comparison of metal accumulation between bags filled with a Sphagnum palustre L. clone or with native Pseudoscleropodium purum Hedw., one of the most used moss species in biomonitoring surveys, was investigated. The test was performed in sites with different atmospheric contamination levels selected in urban, industrial, agricultural and background areas of Italy and Spain. Among the eighteen elements investigated, S. palustre was significantly enriched in 10 elements (Al, Ba, Cr, Cu, Fe, Hg, Pb, Sr, V and Zn), while P. purum was enriched only in 6 elements (Al, Ba, Cu, Hg, Pb and Sr), and had a consistently lower uptake capacity than S. palustre. The clone proved to be more sensitive in terms of metal uptake and showed a better performance as a bioaccumulator, providing a higher accumulation signal and allowing a finer distinction among the different land uses and levels of pollution. The excellent uptake performance of the S. palustre clone compared to the native P. purum and its low and stable baseline elemental content, evidenced in this work, are key features for the improvement of the moss bag approach and its large scale application.

Best options for the exposure of traditional and innovative moss bags: A systematic evaluation in three European countries.

To develop an internationally standardized protocol for the moss bag technique application, the research team participating in the FP7 European project "MOSSclone" focused on the optimization of the moss bags exposure in terms of bag characteristics (shape of the bags, mesh size, weight/surface ratio), duration and height of exposure by comparing traditional moss bags to a new concept bag, "Mossphere". In particular, the effects of each variable on the metal uptake from the air were evaluated by a systematic experimental design carried out in urban, industrial, agricultural and background areas of three European countries with oceanic, Mediterranean and continental climate. The results evidenced that the shape, the mesh size of the bags and the exposure height (in the tested ranges), did not significantly influence the uptake capacity of the transplanted moss. The aspects more affecting the element uptake were represented by the density of the moss inside the bags and the relative ratio between its weight and the surface area of the bag. We found that, the lower the density, the higher the uptake recorded. Moreover, three weeks of exposure were not enough to have a consistent uptake signal in all the environments tested, thus we suggest an exposure period not shorter than 6 weeks, which is appropriate in most situations. The above results were confirmed in all the countries and scenarios tested. The adoption of a shared exposure protocol by the research community is strongly recommended since it is a key aspect to make biomonitoring surveys directly comparable, also in view of its recognition as a monitoring method by the EU legislation.

Accumulation of airborne trace elements in mosses, lichens and synthetic materials exposed at urban monitoring stations: towards a harmonisation of the moss-bag technique.

Mosses, lichens and cellulose filters were exposed for 17 weeks at four urban monitoring stations in Naples (S Italy) to assess the accumulation of airborne Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Ni, Pb, Ti, V, and Zn. In each site, the element accumulation was significantly higher in the moss Hypnum cupressiforme than in the lichen Pseudevernia furfuracea. Acid washed mosses accumulated the highest amount of trace elements, but the differences in element concentrations among the moss samples exposed after water washing and different devitalisation treatments (acid washing, oven drying and water boiling) and between the lichen samples exposed with and without the nylon bag were not statistically significant. The cellulose filters showed the lowest accumulation capability. The reciprocal ordination of sites and exposed materials showed an increasing contamination gradient (especially for Pb, Cu and Zn) from the background site to the trafficked city streets; this pattern was undetectable from PM(10) data recorded by the automatic monitoring devices operating in the four exposure sites. The element profile in exposed materials did not change substantially throughout the urban area and particles of polluted urban soils seem the main source of airborne metals in Naples. Through a comprehensive evaluation of the results from this and previous studies, a protocol is suggested for the moss-bag monitoring of trace element deposition in urban environments.

Influence of exposure sites on trace element enrichment in moss-bags and characterization of particles deposited on the biomonitor surface.

The hypothesis that exposure environment and land use influence element accumulation and particulate size composition in transplants of Hypnum cupressiforme has been tested using moss-bags containing oven-devitalized material. The samples were exposed for three months in ten green sites and ten roadsides in two areas with different land use (A, residential; B, residential/industrial) in the Trieste conurbation (NE Italy). Observations by SEM and EDX-ray microanalysis revealed that particle density was smaller in samples exposed in A than in B, with prevalence of particles containing Al, Ca, Fe and Si, and in good accordance with the element contents measured by acid digestion and ICP-MS. Moss-bags were generally less contaminated in green sites than in roadsides, apparently due to the different enrichment in coarse particles. In both environments, however, the majority of entrapped particles (up to 98.2%) belongs in the inhalable, small size classes (≤PM(10)). The need for careful selection of the exposure sites during the phase of biomonitoring planning is discussed.

Bags with oven-dried moss for the active monitoring of airborne trace elements in urban areas.

To define a harmonized methodology for the use of moss and lichen bags as active monitoring devices of airborne trace elements in urban areas, we evaluated the element accumulation in bags exposed in Naples in different spring weather conditions for 6- and 12-weeks. Three different pre-exposure treatments were applied to moss and lichen materials: water-washing, acid-washing and oven-drying. During the different exposure periods in the Naples urban environment the moss accumulated always higher amounts of elements (except Hg) than lichens and the element accumulation increased during wetter weather and higher PM(10) conditions. The oven pre-treatment did not substantially modify the morphology and element composition of moss and the exposure in bags of this material for 6-weeks was sufficient to detect the pattern of airborne trace elements.

Natural and pre-treatments induced variability in the chemical composition and morphology of lichens and mosses selected for active monitoring of airborne elements.

To enhance the reliability of the moss and lichen transplant technique for active biomonitoring of trace metals in urban environments, we evaluated the natural variability in the chemical composition of the (epilithic and epiphytic) moss Hypnum cupressiforme and the epiphytic lichen Pseudevernia furfuracea from two reference areas in NE Italy. Green shoots of epilithic mosses and lobes of epiphytic lichens from larch branches showed rather homogenous composition and were selected for the exposure in nylon bags. As different physico-chemical pre-treatments are usually applied to selected cryptogamic material before its exposure, we also evaluated the effects of oven-drying at 120 degrees C for 24h, washing in 1N HNO3 solution, and in 0.5% NH4 oxalate solution at 85 degrees C for 15 h on the chemical composition and morphology of water-washed moss shoots and lichen lobes. Pre-treatments remarkably changed the chemical composition of selected materials but not their surface morphology.

Lichen and moss bags as monitoring devices in urban areas. Part I: influence of exposure on sample vitality.

Samples of the lichen Pseudevernia furfuracea (L.) Zopf and the moss Hypnum cupressiforme Hedw. were exposed for 6 weeks in nylon bags in two air pollution monitoring stations in Trieste and Naples (Italy) with different climates and pollution loads to evaluate influence of environmental conditions on sample vitality. This was assessed before and after exposure by transmission electron microscopy observations, K cellular location, and measurements of C, N, S and photosynthetic pigments content, CO2 gas exchange, and chlorophyll fluorescence. Almost all data sets indicate that exposures caused some damage to the species, considerably heavier in the moss, especially in Naples. The two cryptogams differed significantly in accumulation and retention of C, N, and S, the lichen clearly reflecting NO2 availability. The difference in vitality loss was related to the different ecophysiology of the species, because concentrations of phytotoxic pollutants were low during exposure. Critical notes on the analytical techniques are also given.

Lichen and moss bags as monitoring devices in urban areas. Part II: trace element content in living and dead biomonitors and comparison with synthetic materials.

Lobes of the lichen Pseudevernia furfuracea (L.) Zopf and shoots of the moss Hypnum cupressiforme Hedw. were subjected to different treatments (water washing, oven drying, HNO3 washing, NH4-oxalate extraction) to assess the influence of vitality on accumulation efficiency, during a 6-week exposure in bags in two Italian cities, Trieste and Naples. No trend emerged between treatments, in terms of accumulation ability, for major and trace elements. Only water-washed lichens showed an increased C and N content after exposure in both cities. Element concentrations generally reached higher values in mosses than in lichens, especially for Al, Fe, and Zn (both cities), and for Cu, Mg and Na (Naples). Surface development strongly influenced accumulation capacity of the biomonitors. Quartzose and cation exchange filters revealed, on a weight basis, a poor performance. In urban environments, surface interception of atmospheric particulate seems to play a major role in accumulation, irrespective of organism vitality.

Conditioned medium from mixed retinal pigmented epithelium and Müller cell cultures reduces in vitro permeability of retinal vascular endothelial cells.

AIM: To investigate the in vitro effect of laser photocoagulation on blood-retinal barrier permeability. METHODS: Retinal capillary endothelial cells were exposed to supernatants from long term co-cultured cells that were argon laser treated. Endothelial cell permeability was analysed by (1) measurement of transendothelial electrical resistance and (2) equilibration of [(3)H] inulin and [(14)C] albumin across the cell monolayer. RESULTS: Laser photocoagulation of various retinal cells and control ECV304 cells in the lower chamber did not appreciably improve permeability of the endothelial cell monolayer compared with that of unlasered cells. However, medium that was conditioned by mixed retinal pigmented epithelium and Müller cells significantly reduced both inulin (43.2% (SD 6.5%) equilibration in mixed cultures v 59.8% (SD 7.0%) control cells, p<0.05) and albumin (15.1% (SD 3.8%) v 31.1% (SD 6.7%), p<0.05) permeability of the endothelial cell monolayers. A fourfold increase in transendothelial electrical resistance was also seen. CONCLUSIONS: These results are consistent with the hypothesis that interaction of Müller cells with retinal pigmented epithelium induced by laser treatment results in secretion of soluble factor(s), which reduces permeability of retinal vascular endothelium. Identification of these factor(s) may have implications for the clinical treatment of macular oedema secondary to diabetic retinopathy and other diseases.

CO2 exchange of the endolithic lichen Verrucaria baldensis from karst habitats in northern Italy.

CO2 exchange of the endolithic lichen Verrucaria baldensis was measured in the laboratory under different conditions of water content, temperature, light, and CO2 concentration. The species had low CO2 exchange rates (maximum net photosynthesis: c. 0.45 µmol CO2 m-2 s-1; maximum dark respiration: c. 0.3 µmol CO2 m-2 s-1) and a very low light compensation point (7 µmol photons m-2 s-1 at 8°C). The net photosynthesis/respiration quotient reached a maximum at 9-15°C. Photosynthetic activity was affected only after very severe desiccation, when high resaturation respiratory rates were measured. Microclimatic data were recorded under different weather conditions in an abyss of the Trieste Karst (northeast Italy), where the species was particularly abundant. Low photosynthetically active radiation (normally below 40 µmol photons m-2 s-1), very high humidities (over 80%), and low, constant temperatures were measured. Thallus water contents sufficient for CO2 assimilation were often measured in the absence of condensation phenomena.

Gas exchange rates and chlorophyll content of epi- and endolithic lichens from the Trieste Karst (NE Italy).

The photosynthetic activity of calcicolous endo- and epilithic lichens from the Trieste Karst area (NE Italy) was investigated. The data consist of: (a) gas exchange rates of Acrocordia conoidea, Petractis clausa, Rinodina immersa (endolithic), and Aspicilia calcarea (epilithic), recorded at different combinations of temperature, water content and radiation flux values, (b) chlorophyll content values of nine species, and (c)δ13 C values measured in 24 lichen species with different growth-forms. The results show that: (1) the maximum photosynthetic rates of endolithic lichens are rather small, ranging between 0·2 and 1·5µmol CO2 m-2 h-1 at optimal conditions; (2) the relation between thallus water content and photosynthesis differs from that of foliose and fruticose lichens; the optimum water content of endolithic lichens is particularly small, when expressed in g H2 O cm-2 ; (3) the algae of endolithic lichens, which belong to different systematic groups, are light-saturated at a small radiation flux; (4) chlorophyll contents of endolithic lichens are rather large, being similar to those of some parmelioid lichens. The ecological implications of the endolithic growth-form are briefly discussed. Endolithic lichens should be regarded as slow-growing, stress-tolerant organisms, which are rather similar in their physiology to epilithic crustose lichens; they have a high resistance to CO2 diffusion, saturation being reached only at a very large CO2 concentration.