Background element content in the lichen Pseudevernia furfuracea: a comparative analysis of digestion methods.

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.

Genetic structuring of the moss Pseudoscleropodium purum sampled at different distances from a pollution source.

In this study, we used amplified fragment length polymorphism analysis to investigate the genetic structure of the terrestrial moss Pseudoscleropodium purum (Hedw.) M. Fleish. naturally exposed to different levels of atmospheric deposition of heavy metals. We also determined the heavy metal concentrations in samples of this moss to evaluate whether there was a relationship between atmospheric pollution and population genetic diversity. A low level of genetic diversity and a limited gene flow among populations were observed which is in accordance to the prevalence of asexual reproduction in this species. In addition, no significant correlation was found between metal content and gene diversity in P. purum, probably because of the common history of the sampled populations and/or to the lack of a drastic reduction of the size of the population; nonetheless, a clear genetic structure was evident in relation to the existing pollution gradient. Thus, based on the results of the principal coordinate analysis and Bayesian analysis of the genotypes, the mixed structure of the second most polluted population would suggest an ongoing differentiation of metal-tolerant genotypes in the most polluted sites of the sampling area.

Persistent pollutants and the patchiness of urban green areas as drivers of genetic richness in the epiphytic moss Leptodon smithii.

We determined genetic variation and metal and polycyclic aromatic hydrocarbon concentrations in Leptodon smithii moss collected in holm oak stands at cities, outskirts and remote areas of Campania and Tuscany (Italy) to investigate if anthropogenic pressure (pollutant emissions and land use change) affects moss genetic richness. In both regions, metal and polycyclic aromatic hydrocarbon concentrations reflected the trend urban>outskirts>remote areas, excepting Tuscany remote site. In both regions, the moss gene diversity increased from urban to remote areas. The findings suggest the extent and the fragmentation of urban green areas, as drivers of moss genetic richness.

The Potential of Plants to Absorb Xenobiotics.

Environmental pollution is a pressing problem that endangers our biosphere [...].

Phytomonitoring and Phytoremediation of Environmental Pollutants.

Since 1973, with the activation of the Environmental Action Program (EAP), the European Union has produced a substantial body of legislative packages aimed at improving the environmental quality; as a result, air, water, and soil pollution have significantly been reduced, as stated by the European Commission in the EAP 2020 [...].

Seed Priming by Low-Dose Radiation Improves Growth of Lactuca sativa and Valerianella locusta.

Valerian salad and lettuce are edible species that are easy to grow rapidly, and have traits useful for commercial purposes. The consumption of these species is increasing worldwide for their nutritional properties. Seed germination and seedling development are critical stages in the life cycle of plants. Seed priming, including the use of high-energy radiation, is a set of techniques based on the idea that low stress levels stimulate plant responses, thereby improving seed germination and plant growth. In this study, we evaluated in hydroponic culture (i) the germination performance; (ii) morphological traits; and (iii) antioxidant and phenol contents at different endpoints in Lactuca sativa and Valerianella locusta that were developed from seeds exposed to X-rays (1 Gy and 10 Gy doses). Under radiation, biomass production increased in both species, especially in lettuce, where also a reduction in the mean germination time occurred. Radiation increased the level of phenols during the first growth weeks, under both doses for lettuce, and only 1 Gy was required for valerian salad. The species-specific responses observed in this research suggest that the use of radiations in seed priming needs to be customized to the species.

A new green protocol for the identification of microplastics and microfibers in marine sediments, a case study from the Vesuvian Coast, Southern Italy.

Microplastics (MP; 1 µm-5 mm) and microfibers (MF; thin, elongated particles with a high-length-to-width ratio) have become a major global environmental issue due to their ubiquity in the oceans and possess complex physicochemical properties that vary their mobility, bioavailability, and toxicity toward organisms and interactions with their surrounding pollutants. Nonetheless, a reliable methodology that would facilitate and automate the monitoring of MP is still lacking. Intending to select practical and standardized methods and considering the challenges in MPs detection, a new analysis protocol based on optical microscopy for the counting and morphological analysis of the particles has been developed. This method overcomes some issues related to the lack of practicality and standardization of the others currently applied, and does not involve sieving, washing, heating, or density separation and digestion processes. Our method is green and requires a minimum quantity of sediment, i.e., 1.5 g, and shortened timeframes. Future research efforts may need to develop and implement new analytical tools and combinations of technologies to complement respective detection limitations and yield reliable characterization of both MFs and MPs. We tested our protocol to study, for the first time, both marine and land sediment in the Vesuvian area of the Gulf of Naples (Italy).

Evaluation of morpho-physiological responses and genotoxicity in Eruca sativa (Mill.) grown in hydroponics from seeds exposed to X-rays.

Due to its potential applications in cultivated plants, ionizing radiation (IR) and its effect on organisms is increasingly studied. Here we measured the effects of ionizing radiation on Eruca sativa by analyzing plants from irradiated seeds (1 and 10 Gy) grown in hydroponics. We measured several morpho-physiological traits and genotoxicity. Radiation stress induced a noticeable variability of the morpho-physiological traits highlighting decreased plant vigor. Shoot length and leaf number were significantly higher in 1 Gy-treated samples, whereas root length was significantly higher in 10 Gy treated plants. Stomata number significantly increased with IR dose, whereas both pigment and Rubisco content decreased under radiation stress. Phenol content significantly increased in 1 Gy treated samples, otherwise from total antioxidants, which were not different from control. Most results could find a feasible explanation in a hormesis-like pattern and in a decreased plant vigor under radiation stress. IR induced genotoxic damage, evaluated by ISSR markers, in 15 day old leaves; specifically, a severe decrease in the genome template stability was observed. However, a partial recovery occurred after 2 weeks, especially under the lowest dose (i.e., 1 Gy), suggesting that DNA damage detection and repair mechanisms are active. Pigment content and genotoxic damage may serve as proxies for evaluating plant responses to IR stress, since they show univocal dose-dependent trends. The use of more checkpoints for analyses and more doses over a wider range, as well as the focus on different metabolites, could help elucidate plant response in terms of morpho-physiological changes.

Biomonitoring of Airborne Microplastic Deposition in Semi-Natural and Rural Sites Using the Moss Hypnum cupressiforme.

We show that the native moss Hypnum cupressiforme can be used as a biomonitor of atmospheric microplastics (MPs). The moss was collected in seven semi-natural and rural sites in Campania (southern Italy) and was analyzed for the presence of MPs, according to standard protocols. Moss samples from all sites accumulated MPs, with fibers representing the largest fraction of plastic debris. Higher numbers of MPs and longer fibers were recorded in moss samples from sites closer to urbanized areas, likely as the results of a continuous flux from sources. The MP size class distribution showed that small size classes characterized sites having a lower level of MP deposition and a high altitude above sea level.

Optimizing Moss and Lichen Transplants as Biomonitors of Airborne Anthropogenic Microfibers.

Anthropogenic microfibers (mfs) are synthetic particles composed of cellulose (cotton, rayon, acetate, etc.) or petrochemical-based polymers (i.e., microplastics-MPs) that are less than 5 mm in length. The accumulation of mfs, including MPs, in the moss Hypnum cupressiforme and the lichen Pseudevernia furfuracea was compared in a transplant experiment lasting 6 weeks. We also tested the effects of the bag used for transplants on the accumulation of mfs. Anthropogenic particles trapped by both biomonitors were mostly filamentous (99% mfs), and their number was overall higher in the moss (mean ± s.d. 102 ± 24) than in the lichen (mean ± s.d. 87 ± 17), at parity of sample weight. On average, mfs found in lichen were significantly longer than those found in moss bags, suggesting that lichens are less efficient at retaining smaller mfs. Exposure without the net yielded a higher mfs number accumulation in both species, indicating that "naked" transplants provide greater sensitivity. The calculation of daily fluxes evidenced a loss of mfs in the lichen, suggesting the presence of more stable bonds between moss and mfs. Raman microspectroscopy carried out on about 100 debris confirms the anthropogenic nature of mfs, of which 20% were MPs. Overall results indicate that moss is preferable to lichen in the biomonitoring of airborne mfs especially when exposed naked.

Metals Induce Genotoxicity in Three Cardoon Cultivars: Relation to Metal Uptake and Distribution in Extra- and Intracellular Fractions.

Heavy metal-polluted soil represents an important stress condition for plants. Several studies demonstrated that growth inhibition under metal stress and metal-induced damages, including genotoxicity, is particularly pronounced at the early stages of seedling growth. Moreover, it is reported that heavy metals enter the cytoplasm to exert their detrimental effect, including DNA damage. In this work, we estimated (i) metal-induced genotoxicity by ISSR molecular markers and (ii) the distribution of the metal fractions between symplast and apoplast by EDTA washing, in three cultivars of Cynara cardunculus var. altilis (L.) DC (Sardo, Siciliano, and Spagnolo), grown in hydroponics for 15 days with Cd or Pb: In line with the literature, in all cultivars, the genotoxic damage induced by Pb was more severe compared to Cd. However, a cultivar-specific response was evidenced since Spagnolo showed, under metal stress, a significantly higher genome template stability compared to the other examined cultivars. The lower genotoxicity observed in Spagnolo could depend on the lower intracellular metal concentration measured in this cultivar by chemical analysis. Accordingly, light microscopy highlighted that Spagnolo developed smaller and more numerous epidermal cells under metal stress; these cells would provide a larger wall surface offering a wider metal sequestration compartment in the apoplast.

Multi-elemental profile and enviromagnetic analysis of moss transplants exposed indoors and outdoors in Italy and Belgium.

Air pollution represents one of the major concerns worldwide, fueled by the increasing urbanization and related PM production worsening air quality in open air as well as in confined environments. In the present work, exposure to atmospheric metal pollution was investigated in 20 paired indoor (I)-outdoor (O) sites located in two urban areas of Italy and Belgium, by chemical (ICP-MS) and magnetic (saturation isothermal remanent magnetization, SIRM) analyses of Hypnum cupressiforme moss exposed in bags. After 12 weeks, the elemental profiles of the moss material exposed in the two countries largely overlapped, except for some elements which specifically accumulated in Belgium (Ag, As, Cd, Mo, Pb and Sb) and in Italy (Ca, Mg, Co, Cr, Sr, Ti and U). Element concentrations were higher in moss exposed outdoors, with the Italian sites mostly showing a terrigenous footprint, and the Belgian sites mostly affected by elements of environmental concern (e.g., As, Pb, Sb). The Indoor/Outdoor ratios (mostly lower than 0.75) indicated indoor pollution as strongly affected by outdoor pollution, although specific elements could be of indoor origin or magnified in indoor environments (e.g., Al, Ag, Cd and Co). In line with the chemical analysis, the SIRM signal was significantly higher in outdoor than indoor moss material. A positive, significant correlation was observed between SIRM and several accumulated elements indicating SIRM analysis as a powerful tool to predict the level of metal pollution. Moss bags were confirmed as a useful and versatile tool to highlight metal contamination even in confined environments, an essential prerogative in the perspective of the evaluation of the total exposure risk for humans to these pollutants.

Mobile Biomonitoring of Atmospheric Pollution: A New Perspective for the Moss-Bag Approach.

In this work the potential of moving moss-bags, fixed to bicycles, to intercept particulate matter (PM) and linked metal(loid)s was tested for the first time. Seven volunteers carried three moss-bags for fifty days while commuting by bicycle in the urban area of Antwerp, Belgium. Moreover, one bike, equipped with mobile PM samplers, travelled along four routes: urban, industrial, green route and the total path, carrying three moss-bags at each route. The saturation isothermal remanent magnetization (SIRM) signal and chemical composition (assessed by HR-ICP-MS) of the moss samples indicated that the industrial route was the most polluted. Element fluxes (i.e., the ratio between element daily uptake and the specific leaf area) could discriminate among land uses; particularly, they were significantly higher in the industrial route for Ag, As, Cd and Pb; significantly lowest in the green route for As and Pb; and comparable for all accumulated elements along most urban routes. A comparison with a previous experiment carried out in the same study area using similar moss-bags at static exposure points, showed that the element fluxes were significantly higher in the mobile system. Finally, PM2.5 and PM10 masses measured along the four routes were consistent with element fluxes.

Facing metal stress by multiple strategies: morphophysiological responses of cardoon (Cynara cardunculus L.) grown in hydroponics.

The contamination of environments by heavy metals has become an urgent issue causing undesirable accumulations and severe damages to agricultural crops, especially cadmium and lead which are among the most widespread and dangerous metal pollutants worldwide. The selection of proper species is a crucial step in many plant-based restoration approaches; therefore, the aim of the present work was to check for early morphophysiological responsive traits in three cultivars of Cynara cardunculus (Sardo, Siciliano, and Spagnolo), helping to select the best performing cultivar for phytoremediation. For all three tested cultivars, our results indicate that cardoon displays some morphophysiological traits to face Cd and Pb pollution, particularly at the root morphology level, element uptake ability, and photosynthetic pigment content. Other traits show instead a cultivar-specific behavior; in fact, stomata plasticity, photosynthetic pattern, and antioxidant power provide different responses, but only Spagnolo cv. achieves a successful strategy attaining a real resilience to metal stress. The capacity of Spagnolo plants to modify leaf structural and physiological traits under heavy metal contamination to maintain high photosynthetic efficiency should be considered an elective trait for its use in contaminated environments.

Instrumental and bio-monitoring of heavy metal and nanoparticle emissions from diesel engine exhaust in controlled environment.

In the present article we characterized the emissions at the exhaust of a Common Rail (CR) diesel engine, representative of light-duty class, equipped with a catalyzed diesel particulate filter (CDPF) in controlled environment. The downstream exhausts were directly analyzed (for PM, CO, CO2, 02, HCs, NOx) by infrared and electrochemical sensors, and SEM-EDS microscope; heavy metals were chemically analyzed using mosses and lichens in bags, and glass-fibre filters all exposed at the engine exhausts. The highest particle emission value was in the 7-54 nm size range; the peak concentration rose until one order of magnitude for the highest load and speed. Particle composition was mainly carbonaceous, associated to noticeable amounts of Fe and silica fibres. Moreover, the content of Cu, Fe, Na, Ni and Zn in both moss and lichen, and of Al and Cr in moss, was significantly increased. Glass-fibre filters were significantly enriched in Al, B, Ba, Cu, Fe, Na, and Zn. The role of diesel engines as source of carbonaceous nanoparticles has been confirmed, while further investigations in controlled environment are needed to test the catalytic muffler as a possible source of silica fibres considered very hazardous for human health.

Exploring the phytoremediation potential of Cynara cardunculus: a trial on an industrial soil highly contaminated by heavy metals.

Metal uptake capacity and growth patterns of three cardoon cultivars (Sardo, Siciliano, and Spagnolo) were investigated for phytoremediation in a metal-contaminated soil. Metal concentrations after one and two growth rounds were measured in soil and plants by ICP-MS. Potentially and promptly available metal fractions were estimated by EDTA and ammonium nitrate. Plant weight, water and nitrogen content, and life span were considered as growth parameters. After the first growth cycle, a significant accumulation of all elements was observed in roots, whereas only Cd, Cu, Fe, Pb, and Sb were significantly translocated to the shoots. After the second growth cycle, a further significant increase, especially in roots, was observed in all cultivars and for all elements considered. High percentages of the potentially available fraction were found for As, Cd, Cu, Pb, and Sb. Metal concentrations in plants were far higher than the promptly available fraction, indicating an increase of metal bioavailability during culture rounds. A severe decrease of weight and life span was observed in Sardo and Siciliano grown on polluted soil, along with an increase in N content. The cultivar Spagnolo, harmonizing a high ability of metal uptake and tolerance, proved the most efficient cultivar in metal phytoremediation.

Clonal diversity and geographic structure in Pleurochaete squarrosa (Pottiaceae): different sampling scale approach.

This paper describes our investigation of genetic variation and clonal structure of the Mediterranean moss Pleurochaete squarrosa (Brid.) Lindb. (Pottiaceae), using inter-simple sequence repeat (ISSR) molecular markers and trnL(UAA) (intron of plastid gene for Leu tRNA) sequence, choosing different sampling strategies and scales on 16 European populations. Moreover, the intercontinental distribution of two trnL haplotypes, previously found over a large area and including 24 populations in three continents, was also investigated. Despite the prevalent asexual reproduction, P. squarrosa shows a high level of genetic diversity. Some site features seem to affect the clonal structure at the local scale, influencing the relocation of detached fragments and the level of intermingling, but they do not substantially affect genetic diversity. The peculiar vegetative reproduction coupled with somatic mutation could partly account for the genetic variation detected. Genetic distances highlight geographic isolation and limited gene flow among populations. We found only two trnL haplotypes in Europe due to length polymorphism, but, over an intercontinental scale, only non-delete trnL was found in Africa and the USA. ISSR analysis within each population detected a higher genetic distance between the samples with different trnL haplotypes, suggesting the presence of two different genetic lineages within this species, geographically overlapping in the Mediterranean Basin.

Overall plant responses to Cd and Pb metal stress in maize: Growth pattern, ultrastructure, and photosynthetic activity.

This study provides a full description of the responses of the crop energy plant Zea mays to stress induced by Cd and Pb, in view of a possible extensive use in phytoattenuation of metal-polluted soils. In this perspective, (i) the uptake capability in root and shoot, (ii) the changes in growth pattern and cytological traits, and (iii) the photosynthetic efficiency based on photochemistry and the level of key proteins were investigated in hydroponic cultures. Both metals were uptaken by maize, with a translocation factor higher for Cd than Pb, but only Cd-treated plants showed a reduced growth compared to control (i.e., a lower leaf number and a reduced plant height), with a biomass loss up to 40%, at the highest concentration of metal (10-3 M). The observation of cytological traits highlighted ultrastructural damages in the chloroplasts of Cd-treated plants. A decline of Rubisco and D1 was observed in plants under Cd stress, while a relevant increase of the same proteins was found in Pb-treated plants, along with an increase of chlorophyll content. Fluorescent emission measurements indicated that both metals induced an increase of NPQ, but only Cd at the highest concentration determined a significant decline of Fv/Fm. These results indicate a different response of Z. mays to individual metals, with Pb triggering a compensative response and Cd inducing severe morpho-physiological alterations at all investigated levels. Therefore, Z. mays could be successfully exploited in phytoattenuation of Pb-polluted soil, but only at very low concentrations of Cd to avoid severe plant damages and biomass loss.

Background element content of the lichen Pseudevernia furfuracea: A supra-national state of art implemented by novel field data from Italy.

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.

Genotoxic effect of Pb and Cd on in vitro cultures of Sphagnum palustre: An evaluation by ISSR markers.

In the present work, the genotoxic effect of cadmium and lead supplied in a laboratory trial, was investigated for the first time in the moss Sphagnum palustre, by ISSR molecular markers. A total of 169 reproducible bands were obtained with 12 primers, ten of which gave polymorphisms (i.e., appearance/disappearance of bands), indicating a clear genotoxic effect induced by the metals. Both metals induced a decrease of the genome template stability in a dose dependent manner. At concentration >10-5 Cd also induced a general toxic effect in S. palustre, leading to chlorophyll degradation and moss death. Moreover, we followed the fate of supplied heavy metals into the moss tissue by SEM-EDX to see if they entered the cells. SEM-EDX observations on moss cultures treated with equimolar concentrations of the two metals showed that most Pb precipitated in form of particles on moss surface, while Cd did not aggregate in particles and was not found on moss surface. In light of these findings, we concluded that probably Pb induced a genotoxic effect at lower intracellular concentrations than Cd.