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In bioaccumulation studies, sample devitalization through acid washing or oven drying is commonly applied to enhance the element accumulation efficiency of moss sample. Such aspect, however, has never been considered in biomonitoring surveys using lichens. In this study, the trace element accumulation performance of living (L) and dead (D) samples of the lichen Pseudevernia furfuracea was compared by a side-by-side transplanting at 40 sites in a large, mixed land use area of NE Italy for 8 weeks. Devitalization was achieved without any physico-chemical treatments, by storing lichen samples in a dark cool room for 18 months. Health status of lichens was assessed before and after the sample exposure by chlorophyll fluorescence emission. Although elemental analysis of the two exposed sample sets revealed a similar trace element pollution scenario, the content of 13 out of the 24 selected elements was higher in D samples. By expressing results as exposed-to-unexposed (EU) ratio, D samples show a higher bioaccumulation signal in 80% of transplant sites for Al, Ca, Fe, Hg, Pb and Ti. Overall, the health status of lichen samples might lead to interpretational discrepancies when EU ratio is classified according to the recently proposed bioaccumulation scale.

In this study, the effects of ozone (O3) on the physiology of the lichen Pseudevernia furfuracea var. furfuracea previously subjected to field stressing conditions were assessed. Samples collected in a pristine site were exposed for 6 weeks at 3 sites characterized by different pollution, e.g. elemental and PAH depositions (site RU, close to wood-burning house chimneys; site UI, close to cast-ironworks; site CK, in a semi-natural context). Afterwards, samples were transferred to controlled fumigation chambers, where they were either O3-treated for 2 weeks (250 ppb O3 for 5 h day−1, O3+ samples) or not (0 ppb O3, O3− samples). Three physiological markers (Fv/Fm, maximum quantum yield of primary photochemistry; MDA, malondialdehyde content; potassium leakage) as well as elemental and PAH concentrations were measured in matched sets of sample replicates at each experimental step. Data were explored by multivariate techniques and the effects of field exposure and fumigation were tested by generalized linear models (GLM). Detrimental effects on MDA and Fv/Fm were observed limited to samples exposed in RU and UI sites. Physiological parameters in O3-treated samples showed heterogeneous variation patterns with respect to field-exposed ones. A recovery of Fv/Fm was observed in RU- and UI-exposed samples, whereas a significant increase of MDA was highlighted limited to CK O3+ and CK O3− samples, possibly related to a “chamber effect”. Overall, the impairment caused by ozonation was limited, proving the strong O3-tolerance of our test species. Interestingly, the content of the most abundant 4-ring PAHs in RU O3+ samples, which underwent the highest field enrichment of PAHs, was significantly lower than that of matched RU O3− samples. This suggested a possible role of ozone in degrading PAHs at thallus level, with interesting interpretative repercussions in the context of transplant-based surveys aimed at evaluating PAH depositions when O3 ground levels are high.

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.

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.

Considerata l’elevata eterogeneità dell’inquinamento urbano e l’aumento stagionale delle concentrazioni di O3 troposferico, la valutazione degli effetti combinati di diversi inquinanti sulla fisiologia dei licheni assume carattere centrale nell’ottica di un consolidamento metodologico del biomonitoraggio di inquinanti aerodiffusi. In questo lavoro si valutano gli effetti combinati di IPA, elementi e O3 sulla fisiologia di Pseudevernia furfuracea, un biomonitor di amplissimo utilizzo. Essendo inoltre l’ozonazione una tecnica consolidata per la rimozione di IPA da matrici contaminate, a fortiori si ipotizza che l’esposizione dei talli a concentrazioni simil-estive di O3 possa comportare un decremento degli IPA accumulati. I talli sono stati esposti per 6 settimane in siti caratterizzati da diverse fonti emissive e grado di inquinamento. A valle dell’esposizione i campioni sono stati incubati per 2 settimane in camere di fumigazione con o senza O3. Il contenuto di 18 IPA (GC/MS) e 25 elementi (ICP-MS), nonché il contenuto di malondialdeide (MDA), il rilascio di ioni K+ e il parametro Fv/Fm sono stati misurati prima e dopo l’esposizione e la fumigazione. L’effetto dei trattamenti sui parametri fisiologici e sul contenuto di IPA sono stati testati tramite modelli lineari generalizzati (GLM). Sia l’esposizione che la fumigazione hanno avuto effetti statisticamente significativi sul contenuto di MDA ma non su altri marker fisiologici. In particolare Fv/Fm ha subito decrementi significativi rispetto alla condizione pre-esposizione solo a seguito dell’esposizione nei siti più inquinati, così come il rilascio di ioni K+, mentre il contenuto di potassio e fosforo è risultato significativamente inferiore nei talli fumigati rispetto a quelli non fumigati. La concentrazione di IPA a 4 anelli (la classe di IPA più abbondante) è inoltre risultata sistematicamente inferiore nei talli fumigati, confermando un effetto di degradazione di tali composti da parte dell’O3.

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.

The epiphytic lichen Pseudevernia furfuracea is widely used as biomonitor of airborne trace elements and other contaminants and consists of two taxonomic varieties (var. furfuracea and var. ceratea). Here, we assessed the occurrence of inter-varietal differences in the elemental composition of paired samples of var. furfuracea and var. ceratea collected in 20 remote sites of Italian mountains. The concentration of 40 elements was measured by inductively coupled plasma mass spectroscopy, after digestion with HNO3 and aqua regia. The magnitude of inter-varietal differences compared to the effect of large-scale site-dependent environmental factors (i.e., lithological substrate, host tree species, and altitude) on overall element content was explored by multivariate analysis techniques and tested by generalized linear mixed modeling (GLMM). Further GLMMs were separately fitted for each element testing taxonomic-related variability against uncertainty associated to the analytical procedure. Inter-varietal differences were statistically significant only for Hg and P, with higher content in var. ceratea at most sites, and for Mg and Zn, showing the opposite pattern. Since the elemental composition of P. furfuracea in remote sites was mostly affected by local lithology and climatic conditions, our results confirm that lichen material for active biomonitoring should be collected in a single ecologically homogeneous remote area. We also indicate sites in the Eastern Alps where P. furfuracea showed the minimum content of most elements, which are suggested as locations to collect lichen material for transplants. Besides the context-dependency at large spatial scale, variations of elemental composition apparently related to taxonomy, could possibly be due to unequal incidence of morphological traits of the collected material. Further research is needed to clarify this issue, and how it affects bioaccumulation phenomena.