Valeria Spagnuolo

Biodiversity and trace element content of epiphytic bryophytes in urban and extraurban sites of southern Italy

In the present study epiphytic bryophytes were employed as a model system in an integrated way both as bioindicators at species, population and community levels and as bioaccumulators of airborne trace elements. Twenty sites with Quercus ilex trees were chosen in a Mediterranean area both in urban and natural locations, with an altitude ranging from 0 to about 500 m a.s.l., in coastal and inland areas (Campania, Italy). Data on the presence of species, cover values and phenology were recorded for each site and the Index of Atmospheric Purity (IAP) was calculated. The data matrix of frequency/cover × species was analysed by multivariate methods. Data obtained clearly show that in urban sites the number of species and IAP values are lower, and that acrocarpous mosses and vegetative reproduction occur more frequently. Contents of trace elements (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Ti, V, Zn) were measured in four species and concentrations were normalised to the soil pattern by calculating the Enrichment Factor (EF). The results show the large contribution of resuspended soil particles to the chemical composition of the analysed bryophytes. All the examined species were enriched in Cd, Cu and Zn, and in some cases showed high EF for Pb. The differences among epiphytic bryophyte vegetation are discussed in order to evaluate suspected alterations due to human impact and/or environmental change.

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.

Improved biomonitoring of airborne contaminants by combined use of holm oak leaves and epiphytic moss

Concentrations of 12 elements (Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb and Zn) and 16 EPA-listed PAHs were detected in Quercus ilex leaves and the epiphytic moss Leptodon smithii collected at urban, periurban and extraurban holm oak stands, in two Italian Regions (Campania and Tuscany). Levels of environmental contaminants were generally higher in leaves and moss from urban areas than periurban and extraurban ones and samples from Campania had the highest PAH content. The epiphytic moss accumulated higher concentrations of trace elements than leaves and the latter showed a higher accumulation capability for PAHs, especially for those with low molecular weight. The different bioaccumulation in leaves and moss were explained in terms of their distinctive morphological and ecophysiological characteristics. The combined approach seems a promising tool for the monitoring of a wide range of pollutants in Mediterranean urban and extraurban environments.

Ubiquitous genetic diversity in ISSR markers between and within populations of the asexually producing moss Pleurochaete squarrosa

This paper provides an analysis of genetic variability in local populations of the clonal moss Pleurochaete squarrosa, a Mediterranean moss typical of post-fire recovery, and characterised by asexual reproduction. ISSR (Internal Simple Sequence Repeats) primers and trnLUAA (intron of plastid gene for Leu tRNA) length polymorphism were employed to evaluate genetic structure in five southern Italy populations of this moss. Both molecular tools highlight high values of genetic diversity with geographic structure of sampled populations and a low gene flow among the investigated sites. Gene diversity is significant at every hierarchical level of sampling, and generally increases from lower to higher hierarchical levels. Among other factors, the ubiquitous genetic variability detected in P. squarrosa can be related to continuous, occasionally massive, short-range recruitment of propagules, and to the high degree of intermingling, both favoured by the modality of vegetative reproduction and growth occurring in the species.

Cytological stress and element uptake in moss and lichen exposed in bags in urban area.

In this study cytological ultrastructure, total content of C, N and S, and cellular location of major and trace elements (K, Ca, Mg, Cu, Pb and Zn) were investigated in the moss Hypnum cupressiforme and in the lichen Pseudevernia furfuracea exposed in bags for a spring-summer 12-weeks period in the urban area of Naples city. In the moss, severe ultrastructural damages, such as membrane interruptions and dehydration, developed after exposure supporting the occurrence of a dead biomonitor. In the lichen, the post-exposure stress marks, such as the development of lysosome-like vesicles and concentric bodies, or the production of melanin, were overall compatible with life. With exposure, N, S, major and trace element contents all increased in both biomonitors, while C remained substantially unchanged. Copper and Pb were mainly retained in extracellular and particulate forms. Intracellular concentration of Zn consistently increased in both biomonitors, irrespective of their vitality. In transplants, cellular location of elements can better reflect the form in which they occur in the environment.

Molecular biodiversity in the moss Leptodon smithii (Neckeraceae) in relation to habitat disturbance and fragmentation

Bryophytes seem particularly suitable to investigate genetic diversity in relation to habitat disturbance due to their large employment as bioindicators and to the recent application of molecular markers to moss population studies. Genetic variation and structure were analysed in seven urban, extraurban and remote populations of Leptodon smithii, an epiphytic moss of Quercus ilex, a phanerogamic species of Mediterranean climax vegetation. A total of 210 individual shoots were DNA extracted and amplified with internal simple sequence repeat (ISSR) primers, and 54 haplotypes were identified. An uneven distribution of haplotype number and frequencies was observed among sites, with a higher number of haplotypes and more homogeneous haplotype frequencies in the extraurban/remote populations. Molecular diversity indices were overall higher in the extraurban sites than in the urban ones. Multilocus linkage disequilibrium values were in line with the occurrence of sexual/asexual reproduction in the seven populations. The isolation-by-distance model was not supported by Mantel test among sites; however, within-population fixation index (FST) highlighted a clear relation between genetic and physic distances among trees, suggesting a limited dispersal range for L. smithii’s spores. The genetic structure was mainly affected by population size, wood structure and extent, and genetic drift consequent to habitat fragmentation and human-induced disturbance.

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.

Air pollution monitoring using emission inventories combined with the moss bag approach.

Inventory of emission sources and biomonitoring with moss transplants are two different methods to evaluate air pollution. In this study, for the first time, both these approaches were simultaneously applied in five municipalities in Campania (southern Italy), deserving attention for health-oriented interventions as part of a National Interest Priority Site. The pollutants covered by the inventory were CO, NOx, particulate matter (PM10), volatile organic compounds (VOCs), and some heavy metals (As, Cd, Cr, Cu, Hg, Ni, Pb, Se, and Zn). The biomonitoring survey was based on the use of the devitalized moss Hypnum cupressiforme transplanted into bags, following a harmonized protocol. The exposure covered 40 agricultural and urban/residential sites, with half of them located in proximity to roads. The pollutants monitored were Al, As, Cd, Cr, Cu, Fe, Hg, Ni, Pb, Se, and Zn, as well as total polycyclic aromatic hydrocarbons (PAHs) only in five sites. Using the emission inventory approach, high emission loads were detected for all the major air pollutants and the following heavy metals: Cr, Cu, Ni, Pb and Zn, over the entire study area. Arsenic, Pb, and Zn were the elements most accumulated by moss. Total PAH postexposure contents were higher than the preexposure values (~20-50% of initial value). Moss uptakes did not differ substantially among municipalities or within exposure sites. In the five municipalities, a similar spatial pattern was evidenced for Pb by emission inventory and moss accumulation. Both approaches indicated the same most polluted municipality, suggesting their combined use as a valuable resource to reveal contaminants that are not routinely monitored.

Distinguishing metal bioconcentration from particulate matter in moss tissue: testing methods of removing particles attached to the moss surface.

Accurate differentiation of the proportion of bioconcentrated metals (i.e. incorporated into cells) and the proportion that is not bioconcentrated (i.e. adsorbed at the surface) would lead to a better understanding of the uptake processes and would represent an advance in the use of mosses as biomonitors. Traditionally the methods used to remove contaminants that are not bioconcentrated were to wash the plant material with water or to apply the sequential elution technique, but nowadays both options are considered inaccurate for these purposes. The remaining possibilities are to clean the moss samples with a nitrogen jet or by power ultrasound. Samples of terrestrial moss Pseudoscleropodium purum (Hewd.) Fleisch. were collected from five sampling stations. Different nitrogen jet cleaning procedures and ultrasound cleaning procedures were applied to the mosses. To determine whether any of the treatments altered the membrane integrity of the moss samples, the concentrations of K were determined. The shoots were observed under a scanning electron microscope, and the size and number of particles were determined. Nitrogen jet cleaning was determined to be unacceptable because it damaged the phyllids and/or altered the membrane permeability and did not eliminate the particles from the moss surface. Moreover, ultrasound cleaning treatment should also discarded because of the loss of extracellular metals that are transferred to the water in which the moss is cleaned.

Biomonitoring of atmospheric pollution by moss bags: Discriminating urban-rural structure in a fragmented landscape.

In this paper we investigated the possibility to use moss bags to detect pollution inputs - metals, metalloids and polycyclic aromatic hydrocarbons (PAHs) - in sites chosen for their different land use (agricultural, urban/residential scenarios) and proximity to roads (sub-scenarios), in a fragmented conurbation of Campania (southern Italy). We focused on thirty-nine elements including rare earths. For most of them, moss uptake was higher in agricultural than in urban scenarios and in front road sites. Twenty PAHs were analyzed in a subset of agricultural sites; 4- and 5-ringed PAHs were the most abundant, particularly chrysene, fluoranthene and pyrene. Overall results indicated that investigated pollutants have a similar spatial distribution pattern over the entire study area, with road traffic and agricultural practices as the major diffuse pollution sources. Moss bags proved a very sensitive tool, able to discriminate between different land use scenarios and proximity to roads in a mixed rural-urban landscape.