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.

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.

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.

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.

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.

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.

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.

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.

Biosurface properties and lead adsorption in a clone of Sphagnum palustre (Mosses): Towards a unified protocol of biomonitoring of airborne heavy metal pollution.

Although mosses are widely used for active biomonitoring of air pollution, a unified protocol for their treatment before exposure in bags is still lacking. Here we used field- and laboratory-grown Sphagnum palustre L. moss, respectively, treated by EDTA and devitalized by oven drying at 100 °C, to elaborate a consistent procedure of metal and proton adsorption on moss surfaces. Acid-base titrations and Pb2+ adsorption experiments at different pH values and Pb2+ concentrations in solution were performed with both field-collected and laboratory cloned mosses. Devitalization and EDTA treatments did not produce any measurable difference in terms of H+ and Pb2+ adsorption capacities of moss surfaces. The stability constants for Pb2+ adsorption onto moss surfaces as a function of pH (pH-dependent adsorption edge) and at constant pH (5.5 and 6.5) as a function of Pb2+ concentration ("langmuirian" adsorption isotherm) were rather similar between different treatments. A Linear Program Modeling (LPM) of adsorption reactions revealed high similarity of adsorption constants regardless of treatments for both field-grown and cloned mosses. Therefore, in view of the use of S. palustre clone for biomonitoring lead in the environment, we recommend devitalization at 100 °C as unique treatment to perform with the aim to preserve the biomonitor before and after its exposure in bags.

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.

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.

Atmospheric particulate matter intercepted by moss-bags: Relations to moss trace element uptake and land use.

Particulate matter has to be constantly monitored because it is an important atmospheric transport form of potentially harmful contaminants. The cost-effective method of the moss-bags can be employed to evaluate both loads and chemical composition of PM. PM entrapped by the moss Pseudoscleropodium purum exposed in bags in 9 European sites was characterized for number, size and chemical composition by SEM/EDX. Moreover, moss elemental uptake of 53 elements including rare earth elements was estimated by ICP-MS analysis. All above was aimed to find possible relations between PM profile and moss uptake and to find out eventual element markers of the different land use (i.e. agricultural, urban, industrial) of the selected sites. After exposure, about 12,000 particles, mostly within the inhalable fraction, were counted on P. purum leaves; their number generally increased from the agricultural sites to the urban and industrial ones. ICP analysis indicated that twenty-three elements were significantly accumulated by mosses with different element profile according to the various land uses. The PM from agricultural sites were mainly made of natural/crustal elements or derived from rural activities. Industrial-related PM covered a wider range of sources, from those linked to specific industrial activities, to those related to manufacturing processes or use of heavy-duty vehicles. This study indicates a close association between PM amount and moss element-uptake, which increases in parallel with PM amount. Precious metals and REEs may constitute novel markers of air pollution in urban and agricultural sites, respectively.

Infraspecific variability in baseline element composition of the epiphytic lichen Pseudevernia furfuracea in remote areas: implications for biomonitoring of air pollution.

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.

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.

Metal and proton adsorption capacities of natural and cloned Sphagnum mosses.

Terrestrial mosses are commonly used as bioindicators of atmospheric pollution. However, there is a lack of standardization of the biomonitoring preparation technique and the efficiency of metal adsorption by various moss species is poorly known. This is especially true for in vitro-cultivated moss clones, which are promising candidates for a standardized moss-bag technique. We studied the adsorption of copper and zinc on naturally grown Sphagnum peat moss in comparison with in vitro-cultivated Sphagnum palustre samples in order to provide their physico-chemical characterization and to test the possibility of using cloned peat mosses as bioindicators within the protocol of moss-bag technique. We demonstrate that in vitro-grown clones of S. palustre exhibit acid-base properties similar to those of naturally grown Sphagnum samples, whereas the zinc adsorption capacity of the clones is approx. twice higher than that of the samples from the field. At the same time, the field samples adsorbed 30-50% higher amount of Cu(2+) compared to that of the clones. This contrast may be related to fine differences in the bulk chemical composition, specific surface area, morphological features, type and abundance of binding sites at the cell surfaces and in the aqueous solution of natural and cloned Sphagnum. The clones exhibited much lower concentration of most metal pollutants in their tissues relative to the natural samples thus making the former better indicators of low metal loading. Overall, in vitro-produced clones of S. palustre can be considered as an adequate, environmentally benign substitution for protected natural Sphagnum sp. samples to be used in moss-bags for atmospheric monitoring.

Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research.

As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world's climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture (Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S.palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research.