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In bioaccumulation studies, the interpretation of pollutant contents in the target biomonitor has to be performed by assessing a deviation from an unaltered reference condition. A common strategy consists in the comparison with background element content (BEC) values, often built up by uncritically merging methodologically heterogeneous data. In this respect, the acid digestion of samples was identified as a major step affecting BEC data. Here, the analytical outcomes of two acid mixtures were compared on a set of matched paired samples of the lichen Pseudevernia furfuracea, a widely used biomonitor for which BEC values based on partial digestion were previously provided. The standard reference material BCR 482 (P. furfuracea) was used to validate analytical procedures consisting of either a HF total mineralization or an aqua regia partial one, both associated to ICP-MS multi-element analysis. In particular, the performance of the procedures was evaluated by comparing analytical results of field samples with the accuracy obtained on BCR aliquots (measured-to-expected percentage ratio). The total digestion showed a better performance for Al, As, Ba, Ca, Cd, Cu, Fe, Mn, Ni, Se, Sn, and Zn, whereas the opposite was found for Cr, Co, P, and S. Moreover, new BEC values were provided for P. furfuracea using a consolidated statistical approach, after a total sample digestion with hydrofluoric acid. The multivariate investigation of the background variability of 43 elements in 57 remote Italian sites led to the identification of geographically homogeneous areas for which BEC values are provided for use as reference in biomonitoring applications.

Cladina species are likely to suffer the impact of human pressure, resulting in a potential, as well as currently unknown, extinction risk for some of them. In this study, we used herbarium specimen data and literature data combined with geographic information system (GIS)-based analyses to assess the threatened status of Italian Cladina species according to IUCN criteria. A total of 485 records, reported during the period 1833–2013, were evaluated. Biological traits, habitat requirements and distribution patterns were used to infer species extinction risk. Extent of occurrence and area of occupancy have been calculated at the national scale, based on a 2km×2km cell grid. The potential threats for the taxa were assessed using a decision-support protocol in order to set conservation targets for taxa lacking population viability analyses and habitat modelling data. The species were assigned to the IUCN categories mainly using the geographical criterion B, related to species with restricted and fragmented distribution and continuous declining trend, but the species have been tested against the maximum number of criteria for which data were available and/or appropriate. This has provided an opportunity to discuss some basic aspects of the process of lichen red-listing, suggesting some methodological improvements for the mat-forming ones.

Canopy-forming brown algae support highly productive ecosystems whose decline has been attributed to the interplay of several anthropogenic disturbances. Climate change could have disruptive effects on the biology of these species, but the role of temperature in the development of early life stages is poorly understood. The aim of this study was to assess the response of Ericaria giacconei, a winter-reproducing Southern–Mediterranean endemic species, to thermal stress by testing five temperatures (12, 15, 18, 24, and 28°C) on adults and early stages. Chlorophyll a fluorescence of adult plants was measured at 0, 24, 72, and 120 h on nine fronds in each of the three aquaria per treatment. To assess egg release, zygote settlement, and embryo growth rate, approximately 1,200 receptacles were cultured on six Petri dishes per temperature treatment, and 10 random subsections of 2 ×2 mm were examined in three Petri dishes at 0, 20, 44, and 92 h after fertilization. Adult plants showed a plastic physiological response, and thermal stress had no significant effect on PSII efficiency. Embryos fully developed only at 12 and 15°C. Mortality increased at 18 and 24°C, and no zygotes survived at 28°C. In a scenario of further increasing temperatures, the effects of warming could affect the recruitment of E. giacconei and increase its vulnerability to further stresses. These effects on the survival of early stages, which are the bottleneck for the long-term survival of the species, should be taken into account in conservation and restoration measures to maintain canopy-forming macroalgal populations and associated biodiversity and ecosystem services.

In biomonitoring of airborne elements with native lichens, two allopatric species could be used jointly only when bioaccumulation mechanisms are the same, and differences negligible. The present study aims at (i) investigating the bioaccumulation performance for 12 elements in one-year-old lobes of Flavoparmelia caperata and Xanthoria parietina (i.e. the two lichen native biomonitors most used in the peri-Mediterranean regions) collected side-by-side on the same trees at 17 sampling sites. Both species were characterized morphologically and by X-ray diffraction analysis to evaluate specific thallus area and oxalate content, respectively. Results show that Ca and chalcophile elements (Cd, Cu, Pb and Zn) were more concentrated in F. caperata, lithophile and siderophile elements (Cr, Fe, and Ti) in X. parietina, whereas Al, Li, Mn, and Ni were accumulated in similar amounts. The outstanding content of Ca-oxalate deposits in F. caperata underlies the higher content of Ca, Cd and Pb observed in this species. On the contrary, the micro-anatomy of the upper cortex of X. parietina, which is more porous than in F. caperata, allows a higher transfer of litophile and siderophile elements. The adoption of interspecific calibration factors for biomonitoring purposes is thoroughly discussed, and the choice of using a single native species made by the most recent guidelines for biomonitoring airborne elements with lichens is fully supported.

Environmental DNA (eDNA) metabarcoding and metagenomics analyses can improve taxonomic resolution in biodiversity studies. Only recently, these techniques have been applied in aerobiology, to target bacteria, fungi and plants in airborne samples. Here, we present a nine-month aerobiological study applying eDNA metabarcoding in which we analyzed simultaneously airborne diversity and variation of fungi and plants across five locations in North and Central Italy. We correlated species composition with the ecological characteristics of the sites and the seasons. The most abundant taxa among all sites and seasons were the fungal genera Cladosporium, Alternaria, and Epicoccum and the plant genera Brassica, Corylus, Cupressus and Linum, the latter being much more variable among sites. PERMANOVA and indicator species analyses showed that the plant diversity from air samples is significantly correlated with seasons, while that of fungi varied according to the interaction between seasons and sites. The results consolidate the performance of a new eDNA metabarcoding pipeline for the simultaneous amplification and analysis of airborne plant and fungal particles. They also highlight the promising complementarity of this approach with more traditional biomonitoring frameworks and routine reports of air quality provided by environmental agencies.

The metabolic regulations of evergreen Mediterranean Quercus ilex trees growing at extra-urban and urban sites with different percentages of surrounding impervious pavements and distance to major traffic roads were investigated at the peak of the summer drought season. Plants from extra-urban site experienced lower water stress due to a fine regulation of reactive oxygen species (ROS, concomitant rise of anion superoxide and hydrogen peroxide content, +55 and +44%), abscisic acid and water soluble carbohydrates (+111 and +9%). The level of ROS was kept under the injury threshold by both enzymatic and metabolic antioxidants. Plants from the sub-urban site followed a different strategy, with a lower production of ROS and the concomitant reduction in phenols and carotenoids. Despite these differences, indications for stress-induced injury were not observed in any of the sites. Our work shows new relations between urban stress conditions and metabolic regulations of trees growing under such harsh environments.

The interpretation of lichen bioaccumulation data is of paramount importance in environmental forensics and decision-making processes. By implementing basic ideas underlying previous interpretative scales, new dimensionless, species-independent &ldquo, bioaccumulation scales&rdquo, for native and transplanted lichens are proposed. Methodologically consistent element concentration datasets were populated with data from biomonitoring studies relying on native and transplanted lichens. The scale for native lichens was built up by analyzing the distribution of ratios between element concentration data and species-specific background concentration references (B ratios), herein provided for Flavoparmelia caperata and Xanthoria parietina (foliose lichens). The scale for transplants was built up by analyzing the distribution of ratios between element concentration in exposed and unexposed samples (EU ratio) of Evernia prunastri and Pseudevernia furfuracea (fruticose lichens). Both scales consist of five percentile-based classes, namely, &ldquo, Absence of&rdquo, &ldquo, Low&rdquo, Moderate&rdquo, High&rdquo, and &ldquo, Severe&rdquo, bioaccumulation. A comparative analysis of extant interpretative tools showed that previous ones for native lichens suffered from the obsolescence of source data, whereas the previous expert-assessed scale for transplants failed in describing noticeable element concentration variations. The new scales, based on the concept that pollution can be quantified by dimensionless ratios between experimental and benchmark values, overcome most critical points affecting the previous scales.

Melanised and non-melanised thalli of the devitalized lichen biomonitor Pseudevernia furfuracea have higher binding site concentration than mosses, adsorbing 30% more Zn2+, regardless of melanin presence. Despite the broad use of lichens as biomonitors of airborne trace elements, the surface chemistry and metal adsorption parameters of these organisms are still poorly known. The current investigation is aimed at (i) quantifying the acid-base surface properties and the first-order physical-chemical parameters of Cu2+ and Zn2+ adsorption of devitalized Pseudevernia furfuracea, a lichen commonly used in biomonitoring of airborne trace elements, and (ii) comparing the results with those available for moss biomonitors. Equilibrium constants and metal-binding site concentrations were calculated with a thermodynamic model by taking into account the presence/absence of ancillary extracellular cell wall compounds, namely melanin and acetone-soluble lichen substances. An acid–base titration experiment performed in the pH range of 3–10 showed that melanised and non-melanised P. furfuracea samples have lower pHPZC (3.53–3.99) and higher metal-binding site concentrations (0.96–1.20 mmol g−1) compared to that of the mosses investigated so far at the same experimental conditions. Melanin biosynthesis increased the content of carboxyl and phosphoryl groups and reduces that of amine/polyphenols. Cu2+ and Zn2+ adsorption was unaffected by the degree of melanisation while the removal of extracellular lichen substances slightly decreased Zn2+ adsorption. Although Cu2+ and Zn2+ adsorption parameters related to P. furfuracea surfaces were 3 times lower than in the mosses, lichen samples adsorbed the same amount of Cu2+ and 30% more Zn2+. The present study contributes in understanding the role of ancillary cell wall compounds in Cu2+ and Zn2+ adsorption in a model lichen. It also provides a first comparison between the surface physico-chemical characteristics of lichens and mosses frequently used as biomonitors of trace elements.

Products containing graphene-related materials (GRMs) are becoming quite common, raising concerns for environmental safety. GRMs have varying effects on plants, but their impact on the sexual reproduction process is largely unknown. In this study, the effects of few-layer graphene (FLG) and a similarly layered phyllosilicate, muscovite mica (MICA), were tested in vivo on the reproductive structures, i.e., pollen and stigma, of Cucurbita pepo L. ssp. pepo &lsquo, greyzini&rsquo, (summer squash, zucchini). Pollen was exposed to FLG or MICA, after careful physical-chemical characterization, at concentrations of 0.5 and 2 mg of nanomaterial (NM) per g of pollen for up to six hours. Following this, pollen viability was tested. Stigmas were exposed to FLG or MICA for three hours and then analyzed by environmental scanning electron microscopy to verify possible alterations to their surface. Stigmas were then hand-pollinated to verify the effects of the two NMs on pollen adhesion and in vivo pollen germination. FLG and MICA altered neither pollen viability nor the stigmatic surface. However, both NMs equivalently decreased pollen adhesion and in vivo germination compared with untreated stigmas. These effects deserve further attention as they could impact on production of fruits and seeds. Importantly, it was shown that FLG is as safe as a naturally occurring nanomaterial.

In biomonitoring, the knowledge of background element content (BEC) values is an essential pre-requisite for the correct assessment of pollution levels. Here, we estimated the BEC values of a highly performing biomonitor, the epiphytic lichen Pseudevernia furfuracea, by means of a careful review of literature data, integrated by an extensive field survey. Methodologically homogeneous element content datasets, reflecting different exposure conditions across European and extra-European countries, were compiled and comparatively analysed. Element content in samples collected in remote areas was compared to that of potentially enriched samples, testing differences between medians for 25 elements. This analysis confirmed that the former samples were substantially unaffected by anthropogenic contributions, and their metrics were therefore proposed as a first overview at supra-national background level. We also showed that bioaccumulation studies suffer a huge methodological variability. Limited to original field data, we investigated the background variability of 43 elements in 62 remote Italian sites, characterized in GIS environment for anthropization, land use, climate and lithology at different scale resolution. The relationships between selected environmental descriptors and BEC were tested using Principal Component Regression (PCR) modelling. Elemental composition resulted significantly dependent on land use, climate and lithology. In the case of lithogenic elements, regression models correctly reproduced the lichen content throughout the country at randomly selected sites. Further descriptors should be identified only for As, Co, and V. Through a multivariate approach we also identified three geographically homogeneous macro-regions for which specific BECs were provided for use as reference in biomonitoring applications.

In this contribution, new data concerning bryophytes, fungi, and lichens of the Italian flora are presented. It includes new records and confirmations for the bryophyte genera Diplophyllum and Ptychostomum, the fungal genera Arrhenia, Gymnosporangium, and Sporidesmium and the lichen genera Arthonia, Coenogonium, Flavoplaca, Gyalolechia, Parmotrema, Peltigera, Pterygiopsis, Squamarina, Tornabea, and Waynea.

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.

To date, there has been an ever-increasing interest in complementary air monitoring techniques, which may fill the deficiencies of air quality networks. The present work reports the results concerning five biomonitoring surveys (BSs) performed in the proximity of a waste incinerator (WI) over a 16-year period. Hg emission related to the WI activity was monitored by means of both active and passive BSs based on three photosynthetically-active biomonitors (i.e., two epiphytic lichens: Pseudevernia furfuracea and Xanthoria parietina, one vascular plant: Robinia pseudoacacia) collected or exposed before and/or after the WI installation, and after a four-month period of inactivity. Hg concentration values observed in biomonitor samples varied according to the implemented species and to the status of WI plant (active vs. inactive). Our data demonstrate that, in the same pollution scenario, P. furfuracea accumulates three times more Hg than both X. parietina and R. pseudoacacia. The results are discussed in the context of the actual European Union directives concerning air pollution monitoring and assessment, revealing that both active and passive BSs are efficient tools to provide a reliable estimation of the spatial changes of Hg concentrations in the environment.

00002 ăWOS:000388157900039; International audience; This work aimed to define the molecular and chemical signature of a S. palustre clone developed in the framework of the EU-FP7 Mossclone project to improve the standardization and reliability of the moss bag technique. The molecular characterization was performed by a set of DNA molecular markers (RAPD, ISJ, PCR-RFLP, sequencing and microsatellites) to tag the clone produced within the project. Molecular characterization also provided new DNA markers that can be applied in systematic analyses of Sphagnum, and gave new insights to implement well established techniques. The elemental composition of the clone was measured by ICP-MS analysis of 54 major and trace elements, with and without commonly applied pre-exposure treatments (oven devitalization and EDTA washing). Concentrations of almost all analyzed elements were significantly lower (from 10 to 100 times) in the clone than in conspecific field moss, apart from some elements (K, Mo, P and Na) deriving from the culture medium or EDTA treatment. Oven devitalization and EDTA washing did not significantly affect the clone composition. A comparison between the elemental composition of the clone with that of naturally growing Sphagnum species proved the particularly low elemental content of the clone. Therefore, in view of a rigorously standardized moss-bag protocol for the monitoring of persistent atmospheric pollutants, the use of the S. palustre clone, a biomaterial with very low and constant element composition, and homogenous morphological characteristics is strongly recommended. (C) 2016 Elsevier Ltd. All rights reserved.

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.

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 (≤ PM10). The need for careful selection of the exposure sites during the phase of biomonitoring planning is discussed.

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.

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 °C for 24 h, washing in 1 N HNO 3 solution, and in 0.5% NH 4 oxalate solution at 85 °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.

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.

Applications involving graphene-related materials (GRMs) are becoming very common, raising concerns for their environmental impact. GRMs induce various effects on plants, but those on the sexual reproduction are still largely unknown. Here, the effects of graphene oxide (GO) and GO purified from production residues (PGO) were tested in vivo on the stigma of Cucurbita pepo L. ssp. pepo ”Greyzini” (summer squash). Stigmas were exposed to GO or PGO for three hours and were then analyzed by environmental scanning electron microscopy to verify possible alterations to their surface. Stigmas were then hand-pollinated to verify the effects of the two GOs on pollen adhesion and germination on the stigma, and, subsequently, on the development of fruits and seeds. Severe damages to the stigma were not detected; nevertheless, both pollen adhesion and germination on the stigma decreased. Moreover, fruits developed defectively with signs of necrosis in the case of GO, whereas fruits did not ripen in the case of PGO and ovules did not develop seeds after both GOs treatments. These results highlight different mechanisms of interaction of the two materials with the pollen-stigma system, suggesting a possible negative impact of GO on the sexual reproduction of other seed plants.