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Creating and testing a method for controlling the air quality based on a new biotechnological tool. Use of a devitalized moss clone as passive contaminant sensor

Final Report Summary - MOSSCLONE (Creating and testing a method for controlling the air quality based on a new biotechnological tool. Use of a devitalized moss clone as passive contaminant sensor)

Executive Summary:
The MOSSCLONE project (Creating and testing a method for controlling the air quality based on a new biotechnological tool. Use of a devitalized moss clone as passive contaminant sensor) has been a FP7 cooperative project (2012-2015), following the structure of the Grant Agreement nº 282952. According to the project title the aim of the project was to develop and implement a method to control the air quality by using a devitalized moss clone as passive contaminant sensor.
The project has been a highly successful project were all the objectives were completed (including critical tasks) within budget and time. MOSSCLONE has numerous scientific results being generated and, sometimes, being better for monitoring purposes than expected. Some of these results are already published as open access articles but some information is still pending for publication.
Sphagnum palustre was the moss species selected for monitoring purposes and its clone cultivation has been highly optimized using photobioreactors. The exposure and contaminant analysis methods have been established and the clone has been fully characterized. At the end of the project, the method was tested in the surroundings of small scale pollution focus and was also compared with traditional air monitoring techniques.
The created biotool (“mossphere”) has clear competitive advantages over its alternatives, such as cost reduction, lower environmental impact, the higher representativity of its data, the ability to measure numerous pollutants on the same device and the ability to measure pollutants which cannot be monitored with the current technology. At the same time, the commercial value could be supported by the procurement of an European patent (which has been recently applied). Moreover, the wide and frequent dissemination of the project through multiple ways may help to boost its use and/or accept the “mossphere” method within government legislations.

Project Context and Objectives:
European Member States have to deliver periodically precise information about the air quality and the related health within their territories. This information is usually obtained by using monitoring stations. However, the equipment from this stations has several limitations so that, bio-monitoring has been proposed as an adequate alternative.
The MOSSCLONE Project (Creating and testing a method for controlling the air quality based on a new biotechnological tool. Use of a devitalized moss clone as passive contaminant sensor) was a three year EU FP7 cooperative project running from 2012 to 2015, following the structure of the Grant Agreement nº 282952 with the European Commission. It brought together 10 partners from research institutions and small and medium enterprises from across the EU which are shown below:

Participant Nº. Participant legal name Country Organisation type*
1 (Coord) USC Univ. de Santiago de Compostela Spain Academic
2 ALU-FR Albert-Ludwigs Universitaet Freiburg Germany Academic
3 AMRA AMRA - Analisi e Monitoraggio del Rischio Ambientale Scarl Italy Academic
4 UDC Univ. da Coruña Spain Academic
5 CNRS Centre National de la Recherche Scientifique France Academic
6 Biovia Biovia Consultor Ambiental Spain SME
7 Orion Orion Srl Italy SME
8 TecAmb Tecno Ambiente S.L. Spain SME
9 TeLabs T.E Laboratories Ltd Ireland SME
10 MadO Maderas Ornanda Spain SME

The aim of the MOSSCLONE project was to develop and implement a method to control the air quality by using a devitalized moss clone as passive contaminant sensor. Once developed, the method was tested on the field at two European countries (Spain and Italy).
The activities of the MOSSCLONE project were completed successfully within budget and on time. Some experiments were repeated in order to have the full set of samples. However, valuable experience was gained in the development of these experiments, so that, the development of the repeated activities was improved.
The research activities and deliverables from the MOSSCLONE project have been widely disseminated both locally, regionally, nationally and internationally through the publication on scientific journals, oral and poster presentations at conferences and workshops, posts on the project website and social media... As long as we were able, information from the project has been always made available to the public, scientific academia and stakeholders.
And the most important, the MOSSCLONE project has ended with an useful and innovative tool for air quality monitoring which has already aroused the curiosity of different air monitoring stakeholders and, therefore, presents a huge and increasing commercial value. This new biotool (mossphere) has clear competitive advantages over its alternatives, such as cost reduction, a lower environmental impact, the higher representativity of its data, the ability to measure numerous pollutants on the same device/sample and the ability to measure important pollutants which cannot be measured with the current technology. At the same time, the commercial value could be supported by the procurement of a patent at a European scale.

MOSSCLONE has meant the solution to avoid the problems associated with the traditional moss-bag technique to measure air pollution. These problems were: i) the absence of moss species living in urban and industrial reference environments; ii) the availability of natural and unpolluted moss; iii) the natural variability on moss elemental composition; and iv) the absence of a verified and standardized method. Two key studies helped to overcome those problems: i) cultivation in the laboratory of a selected moss clone to always have clean and homogeneous material; and ii) high degree of standardization of the method. The main MOSSCLONE objectives were established and sorted over time with regard to these problems and key studies:
1. Selection of moss species on the basis of existing knowledge about their use as biomonitor and the study of physical-chemical characteristics for the species selected.
2. Creating a pilot bioreactor for the cultivation of the selected species. Based on these results and those of objective 1, one moss species was selected for the isolation and culturing of moss clones.
3. Characterization of the selected moss clone: chemical composition, molecular morphological and physical-chemical characterization.
4. Scaling up moss clone cultivation from pilot bioreactor to large-scale clone production.
5. Design and standardization of moss-bags: type of mesh, shape, and ratio weight moss/size bag.
6. Methodological standardization for exposure conditions: effect of height and exposure time.
7. Moss-bags validation vs. current state-of-the-art methods for air pollution monitoring (bulk deposition, particle samplers, gaseous samplers, and passive samplers). Validation was performed in several different regions, at industrial, urban, and unpolluted environments.
8. To develop a method and perform an initial validation of its usefulness for the detection of atmospheric small scale pollution focus using moss clone bags.


Project Results:
The MOSSCLONE project has successfully completed all the planned tasks. Moreover, the obtained results were even better than expected. For this reason, partners decided to apply for a patent.
Point-by-point and briefly, the project has so far involved:

-The best moss species was selected to be cloned and used: Sphagnum palustre has been selected.
-Sphagnum palustre has been cloned and cultivated on a small and large scale. The cultivation of this species required a special disruption of moss. Moreover, pH values do not need to be controlled during growing.
-The cultivation of the clone has been scaled up and the production has been highly optimized. Each bioreactor (12L) produces about 150 g of dry moss every month.
-The cultivated moss clone has been fully characterized (molecular, physical-chemical and multi-elemental). Each mossphere contains 3 g of cloned moss which is equivalent to 100 m2 of catchment surface. Cloned moss has shown to accumulate more contaminants than native moss.
-Analysis of PAHs in the two species (Pseudoscleropodium purum and S. palustre) has been optimized. Besides, PAHs levels from moss can be determined with only 0.25 g of sample.
-The prototype to contain the moss was specially designed for accumulation purposes and produced on a large scale. The device has been called Mossphere.
-Field sampling was done at three European countries (Spain, Austria and Italy) in order to standardize the moss bag exposure method. The moss bag method has been fully standardized with the obtained information.
-The Mossclone method has been compared with conventional techniques (bulk deposimeters, particle samplers and gaseous samplers). Experiments were done twice and at Spain and Italy. Among other things, Mossphere has shown to be very effective for long term monitoring of Hg.
-Mossclone method has proved to be a cheap, useful and practical technique to detect small scale pollution sources.
-Three full consortium meetings, three WP leaders meetings and three workshops have taken place at Spain, Italy and Germany. The Mossphere was publically presented at the last Workshop which was held at Santiago de Compostela.
-Mossclone project has been spread to the highest level. The project has been mentioned at all types of media (press, conferences, social media, website, radio, Tv...) and different countries.
-Three open access articles have been published with Mossclone results. On the other side, another article has been accepted for publication and more articles will be submitted for publication in the next months.
-With the same environmental burdens, the Life Cycle Assessment of the Mossclone method has shown that representativity of this method is much higher than that from conventional methods.
-Based on all Mossclone results, partners decided to apply for a patent, including the Mossclone method, the moss clone and the Mossphere.

The main studies and results are summarized below according to its Working Package:

WP2 (Moss clone cultivation and characterization)
The WP2 was planned in order to define the cultivation method of the best moss species, a full characterization of the cultivated clone and the analytical optimization of PAHs analysis.
The proper development of this WP was crucial for the project since both critical tasks (cultivation of the clone and analytical optimization of PAHs) of the project were included in this WP.

Task 2.1. Literature review for species selection
As regards the most appropriate species, the information available to date indicates that the most commonly used species are those of the genus Sphagnum (although this genus is absent from several areas). However, in order to have a wider range of possibilities for the cultivation and monitoring tasks our researchers have selected also the six species of moss most used, important (as biomonitor) or available after Sphagnum spp.; Pseudoscleropodium purum, Hypnum cupressiforme, Pleurozium schreberi, Hylocomium splendens, Rhynchostegium riparioides and Brachythecium rutabulum.

Task 2.2. Species selection
Species of the genus Sphagnum best fit the criteria for the suitability of moss species in the moss bags technique. They are of a suitable size for handling and have been cloned, although they have not been cultivated for use in moss bags. The physicochemical properties of Sphagnum also favour their use: the leaves constitute about two thirds of the dry biomass and provide a very large surface area for cation exchange processes and capture of fine airborne particles; the hyalocysts have large pores that can trap airborne particulate; and the cation exchange capacity has been estimated to be much higher than that of other mosses.

Task 2.3. Cultivation of moss clone
Five different moss species were successfully established in axenic in vitro cultures via surface sterilization of spores and gametophores with sodium hypochlorite solutions (Hypnum cupressiforme, Hylocomium splendens, Pseudoscleropodium purum, Rhynchostegium riparioides and the peat moss Sphagnum palustre) and were grown under standardized conditions in the laboratory. In addition, two clones of Brachythecium rutabulum and Rhynchostegium murale (Beike et al. 2010) were cultivated axenically. For Hypnum cupressiforme, Pseudoscleropodium purum, Rhynchostegium riparioides and Sphagnum palustre different independent clones were cultivated in parallel.
Regarding the cultivation procedure, each of the established moss cultures seemed well suited for large-scale cultivation. However, according to the physicochemical characterization of different mosses by our partners, Sphagnum sp. and Hypnum sp. were selected as most suitable for biomonitoring.
In order to establish stable and scalable cultures, Sphagnum palustre was cultivated using different Knop mediums supplemented with microelements. For optimal growth and highest biomass increase, S. palustre was cultivated in Knop medium with microelements supplemented with 1.25 mM ammonium nitrate and 2 % sucrose.
S. palustre has been cultivated with different techniques. We were able to cultivate Gametophores of this species on solid medium in Petri dishes, in Erlenmeyer flasks, aerated round-bottom flasks and in photobioreactors. For large-scale and standardized cultivation, the use of bioreactors was the method of choice.
The cultivation of S. palustre has been highly optimized and scaled up using photobioreactors as shown in Deliverable D3.5.

Task 2.4. Molecular characterization of the moss clone
Several experimental procedures have been carried out but they often failed to produce suitable markers for clone characterization due to the DNA homogeneity of Sphagnum. Anyhow, final molecular characterization of the moss clone was carried out by sequencing (3 regions), SSRs (15 loci), RFLP-PCR of the anonymous DNA region RAPDf (7 polymorphic bands), and ISJs (7 clone-specific loci).
Morphological studies of the clone were also done in this activity. Clone shoots are slender, fragile and lighter than native shoots. The ratio between hyalocyst and chlorocyst wideness is higher in the clone, whereas pores larger and more numerous were observed in native shoots. Cloned shoots showed some breaks and fractures of the cell wall, particularly in the hyalocysts, after EDTA washing protocol and devitalization.
Further information about this activity can be found in deliverable D2.5.

Task 2.5. Analytical optimization (Critical task)
The PAH analysis procedure in moss samples (because the initial selected moss species from these tests was Pseudoscleropodium purum), especially with Sphagnum sp., has been highly optimized.
Several studies have been done during this activity: i) State of the art in the analysis of PAHs in moss (the majority of articles used Hypnum sp. and the Soxhlet technique; ii) Look for drawbacks of the previous method (less co-extraction of interfering substances, perform the evaporation of 12 samples simultaneously, better results for most volatile PAHs, less consumption of sorbents and solvent and a less laborious method); iii) Optimization and Improvements of the method (less consumption of sorbents and solvents, cost reduction, less laborious, shortening of the analysis time); and iv) Study of alternative analytical procedures.
The findings obtained in the different subtasks allowed us to develop an optimized and validated procedure for the analysis of PAHs in moss using microwave assisted extraction. The proposed method improved considerably the initial procedure according to the principles of green chemistry: less consumption of toxic solvents, automation, less laborious and time consuming. The matrix solid-phase dispersion (MSPD) experiments provided great results.
Only 0.25 g of moss is required for the determination of PAHs. The procedure has accuracy between 83 and 112%, and the relative standard deviation (precision) is lower than 11%. Moreover, the detection limits of the method are incredibly low (0.09-1.72 ng g-1). In addition, the performance time has been reduced from one day and a half to 5 hours and the volume of solvent has been reduced to half of the volume (20 mL).
A research paper entitled “Matrix solid phase dispersion: a green procedure for polycyclic aromatic hydrocarbons analysis in moss” has been submitted for open access publishing to Journal of Chromatography A.

Task 2.6. Multi-element characterization of moss clone
Multielemental analysis was carried out on S. palustre clones produced at University of Freiburg (lines 2a and 12a) and at Biovia (line 12a). S. palustre clones produced by Biovia were also analyzed after each of the following pre-treatments:
1) Oven drying at 40 °C for 8 h.
2) EDTA/water washing and drying at 40 °C for 8 h. The washing protocol was as follows: 1 wash of 20 min with 10 mM EDTA (1 L EDTA/12.5 g d.w. moss) and 3 washes of 20 min each with distilled water (1 L distilled water/10 g d.w.)
3) EDTA/water washing and oven devitalization. Three consecutive drying cycles of 8 hours each at 50, 80 and 100 °C were performed for moss devitalization.
S. palustre clones are characterized by very low levels of the studied elements, except for Al, Fe, Mn and Zn. The concentrations of 11 determined elements (i.e. As, Be, Cd, Co, Hg, Pb, Pd, Pt, Rh, Sn and V) were below the detection limits, whereas high levels of Ca, K, Mg, Na, P and S were detected. The high levels of B, Cu, Fe, Mn, Mo and Zn detected in both Freiburg and Biovia untreated clones may come from the clone cultivation medium. More investigations are needed to explain the presence of Al, Ba, Cr, Ni and Sr in all clone samples because these elements were not added in the clone culture medium.
The treatments implemented on Biovia clones determined a decrease of most element concentrations, except for C, Ba and Na. The high Na content could be explained by the use of a disodium EDTA salt. Apparently, the EDTA treatment reduced Hg and N content in Biovia clones. No variations of Mg content were detected after each treatment.
Significant differences were only found when comparing both untreated and treated clones.
Further information about this activity can be found in deliverable D2.6.

Task 2.7. Physical-chemical characterization of moss clone
Four moss species (Hypnum cupressiforme, Sphagnum sp., P. purum and Brachytecium rutabulum) were physical-chemical characterized to consider their potential use as biomonitors of atmospheric pollution. The adsorption of 5 heavy metals (i.e. Cu2+, Cd2+, Ni2+, Pb2+ and Zn2+) was done with these mosses. Solid and solution analyses, including surface acid–base titration, pH-dependent adsorption edge and ‘‘Langmuirian’’ adsorption at constant pH and variable metal concentration, have demonstrated that Sphagnum sp. exhibits the highest proton and metal adsorption capacity while being most stable in aqueous solution in terms of Dissolved Organic Carbon (DOC) release and biomass degradation. The relative difference from other mosses is within 20% despite the species and geographic differences.
The pKa computed for all the moss species suggested the presence of 5 major functional groups: phosphodiester, carboxyl, phosphoryl, amine and polyphenols. The quantitative comparison of metal adsorption with other common natural organic and inorganic materials demonstrates that mosses are among the most efficient natural adsorbents of heavy metals. Mosses possess significantly higher concentration of surface groups capable to bind divalent metals at the cell surface than other bio-sorbents.
Further information about this activity can be found in deliverable D2.7 and in the following published scientific article (open access):
Gonzalez, A.G. Pokrovsky, O.S. 2014. Metal adsorption on mosses: Toward a universal adsorption model. Journal of Colloid and Interface Science 415: 169–178.

WP3 (Tool development)
The main objective of this WP was to standardize the exposure method and setup the new (prototype) tool for monitoring air pollution. For this purpose, two kinds of activities were carried out; firstly, it was necessary to optimize and standardize the way in which the moss clone bags must be exposed. Different aspects have been extensively studied: i) mesh effect; ii) the shape, size and weight of moss effect; iii) the optimal exposure height; and iv) the optimal length of the exposure period. Secondly, and following the results obtained in previous tasks, clone moss bags were produced.

Task 3.1. Moss bag preparation
The manufacturing of huge quantities of moss and different exposure devices has been done. These devices included: flat, rounded, Mossphere and disks. A total of 957 moss containers with native P. purum inside were produced in order to develop the standardization of the method (WP3, Tool development). Then, S. palustre was produced at a large scale in bioreactors and introduced in Mosspheres and disks to be used in WP4 activities (Comparison with traditional methods). A total of 796 moss samples were produced for the latter development of WP4. Approximately, 1760 moss samples were produced during Mossclone project development, some of them were handmade produced (e.g. flat bags, rounded bags and disks) while the Mosspheres were manufactured at a factory using several and specially designed moulds.

Task 3.2. Standardization assays: mesh effect
The accumulation capacity of moss was tested using three different mesh sizes: 1, 2 and 4 mm and native P. purum. The studies were conducted in Austria, Italy and Spain. In each country, seven stations were selected in four scenarios presenting different levels and types of contamination: urban, industrial, agricultural and background.
The concentration of 19 elements was determined in exposed and unexposed moss. Several elements were below the detection limit in unexposed moss samples, therefore, the elements which were used for the comparisons between mesh sizes were Al, Ba, Cr, Cu, Fe, Pb, Ni, Sr and Zn.
The 4 mm mesh seemed to allow a better performance of the moss material. However, based on developed statistical studies, the mesh size does not affect the accumulation of elements in moss in a significant way. The selection of the proper mesh size must thus take into account only the loss of material occurred during exposure. In this sense, the 2 mm mesh has been selected.
Further information about this activity can be found in deliverable D3.1.

Task 3.3. Standardization assays: shape and moss weight effect
Just like the previous activity the accumulation capacity of native P. purum was tested. In this case, three different exposure device shapes (flat, rounded and Mossphere) and moss contents (15, 30 and 45 mg/cm2) were compared in this activity. The studies were also conducted in Austria, Italy and Spain. In each country, seven stations were selected in four scenarios presenting different levels and types of contamination: urban, industrial, agricultural and background.
Flat and rounded bags were the commonly used devices for moss exposure. The spherical Mossphere was specially designed and built within the Mossclone project conforming to our own, new and innovative design. The Mossphere was built in order to be reused several times and to have the maximum exposed surface of moss.
Like the previous activity the concentration of 19 elements was determined in exposed and unexposed moss. However, several elements were below the detection limit.
The Mossphere seems to be less performant when compared to the rounded one, but shows a similar performance with respect to the flat one. In any case, the Mossphere might be preferred for its assemblage readiness, its reusability, and because it might secure an improved standardization of bag preparation, that otherwise would remain a home-made operation.
As far as the quantity of moss material is concerned, the 15 mg/cm2 Mosspheres were found to be significantly more efficient than the 30 and 45 mg/cm2 Mosspheres, having higher enrichments of most of the elements.
Further information about this activity can be found in deliverable D3.2.

Task 3.4. Standardization assays: height effect
The effect of the exposure height on moss accumulation was tested in this activity using three heights: 4, 7 and 10 m. Mosspheres were exposed at the same places than previous standardization studies and the same 19 elements were determined.
Carbon poles of 10 m were specially designed and manufactured for development of this activity.
The height range investigated (4-10 m) did not show any pattern related to the enrichment of post-exposed mosses. In this way, the selection of the exposure height may depend on practical questions (e.g. avoid vandalism) or specific objectives from the study. In order to standardize the method, the Mosspheres will be exposed at 4 m high, which is the most practical height.
Further information about this activity can be found in deliverable D3.3.

Task 3.5. Standardization assays: exposure time effect
The purpose of this activity was to determine the optimal exposure period that should be used for the standardization of the moss bag method. The exposure period must assure the net enrichment in contaminant concentrations on moss during the exposure time and, at the same time, low variability of results. This activity was developed in accordance with previous standardization tasks but, in this case, three different exposure periods (i.e. 3, 6 and 12 weeks) were tested.
The 3 weeks exposure period was not adequate because in most of cases this period is too short to have detectable enrichment in post-exposed mosses. The other two possibilities, 6 or 12 weeks, have both enough sensibility. Regarding to the results replicability, there were not important differences detected among exposure periods. Finally, the uptake rate was higher in 6 and 12 weeks periods than in 3 weeks. As the differences between 6 and 12 weeks exposures were scarce and taking into account operational criteria (i.e. increase temporal resolution), it is recommended to use a 6 weeks exposure period.
Further information about this activity can be found in deliverable D3.4.

Task 3.6. Scaling up to large-scale clone production
In vitro cultures of mosses selected within task 2.1 (literature review for species selection) were obtained (S. palustre, Rhynchostegium murale, B. rutabulum, Hylocomium splendens, P. purum and Hypnum cupressiforme). However, S. palustre was selected according to the results obtained on the physical-chemical characterization for the species selection (task 2.2). For this reason, cultivation studies were continued just with this species.
Axenic cultures of S. palustre were established by following these steps: 1) sterilization from spores in capsules; 2) germination of spores; 3) isolation of single protonema; and 4) cultivation of independent clones.
This task included the performance of several studies regarding S. palustre cultivation: i) the optimal growth medium has been created; ii) different cultivation techniques have been established; and iii) the most suitable pH and tissue disruption have been determined.
Gametophores of S. palustre were cultivated on solid medium in Petri dishes, in Erlenmeyer flasks, aerated round-bottom flasks and in photobioreactors. For large-scale and standardized cultivation, the use of bioreactors was the method of choice.
A bottle with screws inside has been designed to slightly disrupt S. palustre because traditional disruption with Ultraturrax is lethal for this moss. In general, pH values from cultivation medium of mosses are adjusted. However, S. palustre is known to acidify its surrounding habitat and cultivation of this species was developed much better without pH adjustment using a starting pH of 4.
Once the cultivation method was completely optimized BIOVIA bioreactors produced large quantities of cloned moss which were used for the development of WP4 activities.
Additional studies were done about the establishment of a dry weight measurement method and long term storage in cryopreservation. S. palustre was revived living after more than 2 months (76 days) of cryopreservation in -180 ºC.
Further information about this activity can be found in deliverable D3.5. Besides, two articles have been published (open access) about moss cultivation:
Anna K. Beike, Carsten Jaeger, Felix Zink, Eva L. Decker, Ralf Reski, 2014. High contents of very long-chain polyunsaturated fatty acids in different moss species, Plant Cell Reports 33(2): 245-254.
Anna K. Beike, Valeria Spagnuolo, Volker Lüth, Feray Steinhart, Julia Ramos-Gómez, Matthias Krebs, Paola Adamo, Ana Isabel Rey-Asensio, J. Ángel Fernández, Simonetta Giordano, Eva L. Decker, Ralf Reski, 2015. Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research, Plant Cell, Tissue and Organ Culture (PCTOC) 120(3): 1037-1049.

Task 3.7. Clone moss-bags preparation
This activity involved the manufacturing of huge quantities of moss and different exposure devices. These devices included: flat, rounded, Mossphere and disks. A total of 957 moss containers with native P. purum inside were produced in order to develop the standardization of the method (WP3, Tool development). Then, S. palustre was produced at a large scale in bioreactors and introduced in Mosspheres and disks to be used in WP4 activities (Comparison with traditional methods). A total of 796 moss samples were produced for the latter development of WP4. Approximately, 1760 moss samples were produced during Mossclone project development, some of them were handmade produced (e.g. flat bags, rounded bags and disks) while the Mosspheres were manufactured at a factory using specially designed moulds.
Sometimes more than 25 samples were exposed at several sampling sites and during the same exposure period.

WP4 (Detectors)
The aim of WP4 was to compare the results obtained using moss clone bags with those of traditional techniques for monitoring heavy metals, metalloids and PAHs present in the atmosphere. The WP consists in different tasks: i) study the relationship between the pollutants adsorbed by moss clone bags and those collected in bulk deposition, by means of direct comparisons of these two techniques; ii) analyze the relationship between pollutants collected by moss clone bags and those present in particles retained in particles samplers; and iii) study the relationship between data from moss clone bags and those from automatic gaseous samplers.
A second objective was to create a method for detecting atmospheric small scale pollution sources using the Mossphere, since this type of sources of atmospheric pollution are difficult to identify with conventional techniques.
During the first reporting period we found out that the expected quantity of cloned moss required to carry out the planned WP4 was more than we could afford, even more when several WP4 experiments needed to be repeated. The cultivation method has been optimized to the maximum; bioreactors produce in 4 weeks what natural growth would take around three years (each 12 L bioreactor produces about 150 g of dry moss every month). Nevertheless, we have had to reduce the number of samples for WP4 activities, but the number of samples is still big enough to obtain good statistical reliability.
WP4 activities finished just at the end of the project, although studies with data from these tasks will continue to have more robust and detailed results and to be published in open access scientific journals.

Task 4.1. Relationship between standard moss clone bags and bulk deposition
Mossphere samples with cloned S. palustre were exposed together with bulk deposition collectors for 6 weeks in 10 locations from different Italian (6 sites) and Spanish (4 sites) areas; industrial, urban, agricultural and background, in order to study the relationship in the accumulation of PAHs and metals between both methods. The experiments were performed twice at each site and country. The main meteorological parameters (i.e. air temperature, wind speed and direction, atmospheric pressure and precipitation) were registered during exposure periods.
The analyzed PAHs included, in terms of volatility: light PAHs (naphthalene, acenaphthylene, acenaphthene, fluorene), intermediate PAHs (phenanthrene, anthracene, fluoranthene, pyrene) and heavy PAHs (benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(j)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(ghi)perylene and indene(1,2,3-cd)pyrene). On the other side, the heavy metals and metalloids that were studied for this task were: Al, As, Ba, Be, Cd, Cr, Co, Cu, Fe, Hg, Ni, Pd, Pb, Pt, Rh, Se, Sn , Sr, V and Zn.
The PAH profiles obtained with moss samples were very similar to those obtained with the total deposition. This similarity allowed establishing relations between the different PAH and provided information about the possible sources of PAHs pollution. Moss clone Mossphere, as well as traditional methods, performed a good discrimination between the different polluted and clean areas with regard to PAH profiles.
Positive correlations were only found with 4 elements (i.e. Al, Ba, Cu and Sr) collected by deposimeters and the unsheltered Mossphere. Further statistical studies on the relation between the accumulations of metals from both methods are required.
These studies will continue although the project is already over because the activity was finished at the same time as the project.
More information about this activity can be found in deliverable D4.1.

Task 4.2. Relationship between sheltered moss clone bags and particle samplers
Sheltered Mosspheres (covered with a plastic bowl) with cloned S. palustre were exposed together with high and low volume particle samplers at the same sites and conditions than tasks 4.1 and 4.3. The concentrations of the same PAHs, heavy metals and metalloids from the previous task were determined in the suspended atmospheric particulate matter with a diameter lower than 10 µm (PM10).
In this study, sheltered Mossphere collected a higher proportion of light PAHs than the PM10 traditional sampler. The sheltered device hampers the uptake of particulate matter by the moss so it reflects a lower proportion of heavy PAHs than the PM10 traditional sampler. Sheltered Mosspheres, as well as traditional methods, have performed a good discrimination between the different polluted and clean areas with regard to PAH profiles.
Only one significant positive correlation has been found between Ba accumulated by PM10 and sheltered Mossphere. Nevertheless, it is necessary to take into account that the measurement units are not in the same scale (μg/g for moss and ng/m3 for PM10), therefore, the quantitative information about metal concentrations from both methods was not comparable.
These studies will continue although the project is already over because the activity was finished at the same time as the project.
More information about this activity can be found in deliverable D4.2.

Task 4.3. Relationship between sheltered moss clone bags with diffusers and gaseous samplers
Fully sheltered disk bags (covered with a stainless steel “bell”) with cloned S. palustre were exposed together with polyurethane foams (PUF) disks, used as passive samplers (13.5 mm x 140 mm) and also covered with the same stainless steel “bell”. These devices were exposed at the same places and conditions than tasks 4.1 and 4.3. However, only the relations on the accumulation of PAHs were determined when using these devices.
A good discrimination between the different polluted and clean areas was obtained when using the fully sheltered moss bags. The sheltered moss collected less light and intermediate PAHs than the PUF device. On the contrary, heavy PAHs were collected in the moss and almost not detected in the PUF, since the last does not collect the particulate matter.
Hg has been listed as one of the top ten chemicals of public health concern but men has been aware of its toxicity since ancient times. The high spatial and temporal variability in Hg° concentrations makes long-term or large-scale surveys with measuring devices very expensive and almost impossible.
In the framework of the Mossclone project, Mosspheres and commercial SKC Hg passive samplers were exposed together in an abandoned Hg mining and Hg smelting plant at Abbadia San Salvatore (Siena, Italy) to evaluate their relative performances as Hg° accumulators. Three sites were selected inside the abandoned mine with increasing Hg° concentrations.
The uptake of atmospheric Hg was much more rapid in the SKC 520 Series Inorganic Hg Sampler than in the moss clone. However, this sampler was conceived to measure worker exposure levels and it seems unsuitable for field surveys. In open or semi-open environments the Hg accumulation was not linear and values did not reflect the pattern of atmospheric Hg concentrations usually measured in the different sites.
The exposed Mosspheres showed a slower Hg accumulation rate than the sorbent but they behave as long-term integrators of atmospheric Hg. The Hg uptake in the Mossphere was less affected by wind, temperatures or humidity and the amounts of accumulated Hg reflected the pattern of measured Hg° concentrations in the different exposure sites.
Owing to the high spatial and temporal variability in atmospheric Hg concentrations the Mossspheres seem a much more suitable tool to make long-term or large-scale atmospheric Hg° surveys in urban and industrial areas than passive sorbents or other expensive measuring devices.
Further information about this activity can be found in deliverable D4.3

Task 4.4. To develop a method to detect atmospheric small scale pollution focus using moss clone bags
Independent of the obtained results from tasks 4.1 4.2 and 4.3 the present task aimed to develop a method using Mosspheres for the rapid and economic detection of small scale sources of air pollution by heavy metals, metalloids and PAHs. These substances are relevant to detect, because of the high potential toxicity and great environmental persistence of some of these compounds. In order to adapt the methodology previously proposed for native mosses to the use of the Mossphere with cloned moss this task was divided in two main subtasks:
Study of reference normal distributions of the differences in concentration between pairs of sampling sites from unpolluted areas, each separated by a distance of 1 km.
The method for detection of small-scale pollution focus using native mosses is based on the decrease in the concentration of pollutants with increasing distance from emission sources. In a few words, this study verifies that differences in the concentration of elements between moss from the immediate surroundings of the source and another collected at a higher distance from the source are caused by the presence of a contamination source and not simply to variability of data.
The former method allows the rapid and economic detection of small scale sources of air pollution, is extremely useful, and has been recently used with optimal results to obtain an inventory of pollution focuses at a regional scale. Nevertheless, the method was based on native mosses so its use was restricted to environments where these organisms grow naturally. This fact impedes to use this method in highly populated and industrialized areas and areas where mosses are scarce due to climatic conditions. In these cases, the use of moss bags is the only option. Besides, Mosspheres are already available and its use seems to be the most standardized and, as a consequence, logical approach.
Pairs of sampling sites (each separated by a distance of 1 km) were located at national parks or hunting preserves because these places are large enough to hang all the Mosspheres, are far away from industrialized areas and are generally protected and under surveillance.
The performed experiments allowed obtaining reference distributions of differences in concentration between Mosspheres (exposed for 6 weeks at 4 m height) for some heavy metals (i.e. Al, Ba, Cr, Cu, Fe, Hg, Ni, Sr and Zn) and some PAHs (i.e. naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, retene, benzo(a)anthracene, chrysene, benzo(b+j)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, indene(1,2,3-cd)pyrene, and benzo(ghi)perylene).
The uncertainty associated to the exposure of the 3 Mosspheres was, in general, incredibly low for heavy metals. In most cases their values were below 10%, which are noticeable lower than those previously found with native Sphagnum denticulatum (Cu, Hg and Zn uncertainty values doubled those obtained with cloned moss).
Testing the method for detection of small-scale pollution focus using ‘Mosspheres’.
The reference distributions obtained in the previous Task 4.4.1 were used to evaluate if the differences in the concentrations of pollutants from sites close to a small scale pollution source were related or not to natural variability. If the detected differences belonged to those distributions, then, the factory is not emitting pollutants.
Different industries (n=15) were selected in Italy and Spain to evaluate the performance of the method. Three sampling points were selected in the surroundings of every industry; one as close as possible to the factory, the second one at a distance around 1 km from the first, and a third one located also at ca. 1 km from the first site but perpendicularly oriented to the theoretical axis that connects the first and the second site. The distance between the first site and the second either the third was approximately 1 km. Mosspheres were exposed in triplicate according with the standardized conditions (6 weeks and 4 m height).
Only 4 industries out of 15 have been identified as non-emitting pollutants (nor heavy metals nor PAH). Furthermore, the obtained Q.L. values allowed identifying which elements could be used with the method: Al, Ba, Cr, Cu, Fe, Hg, Pb, Ni, Sr, V and Zn. In the case of PAHs all the determinations could be accepted, except for acenaphthylene and dibenzo(a,h)anthracene.
Further information about this activity can be found in deliverables D4.4 and D4.5.

Potential Impact:
1. Impacts
1.1. Call-specific impacts
The call demands proposals on bio-mimetic technologies capable of deriving from nature’s way of functioning. The MOSSCLONE moss-bags make use of nature’s architecture of moss leaves, and their adsorptive capacities. These capacities have shown to be better in the cultivated clone than in native mosses.
Moreover, MOSSCLONE developments offer clear market opportunities for SMEs, as evidenced by the recent application for a patent.

1.2. Research and Development impact
The results of MOSSCLONE will boost basic research and eco-innovation for the coming decades on two main fields:
Selection and generation of moss clones
Studies from culturing and characterizing tasks will enrich our basic knowledge of mosses. Findings will feed research leading to the development of new clones, evolution of moss-bioreactor culture conditions, and the analysis of new moss species.
MOSSCLONE moss-bags for monitoring air pollution
The availability of higher quality data on air pollution will allow the comparison of the results with epidemiological data.

1.3. Economic impact
Affordable and reliable monitoring tools are required to properly support the much needed changes in business culture.
MOSSCLONE technology will be able to support the development of new, eco-friendly products and production processes.
Opportunities for exploitation
The opportunities for exploitation of the moss clone bags are large since the present project has many advantages when compared to state-of-the-art monitoring tools; 1) the new monitoring tool is standardized and can be used worldwide, 2) will reduce the monitoring costs associated with traditional methods and 3) will increase the number of elements that can be analyzed.
A positive result from the patent application will add an extra opportunity for exploitation, especially when considering the applicability, practicality and facility of the method.

1.4. Impact on legislation/ technical standards/ guidelines
In an attempt to comply with the directives 85/337/EEC, 96/62/EC, 2008/50/EC and 2008/1/EC, member states use monitoring stations in urban and peri-urban environments. However, the methods used are usually specific for few gaseous pollutants control and for fine and suspended particulate matter. However, for other pollutants included in the Directives, such as Pb, Cd, As, Ni, Hg and PAHs, analysis on air is too expensive, and their detection is hampered by technical difficulties. This fact is sabotaging the compliance to fore mentioned directives.
The MOSSCLONE moss-bags eliminates all the technical problems from traditional techniques, as i) no energy source is required (allowing to also monitor in remote areas), ii) costs are low (allowing intensive sampling), and iii) a wide range of adsorbed pollutants can be detected and quantified.
Some recent advances have been done regarding policy making. A new norm which considers the use of native mosses for air biomonitoring has been published on February (2014): EN 16414: Ambient air - Biomonitoring with mosses - Accumulation of atmospheric contaminants in mosses collected in situ: from the collection to the preparation of samples.
The Mossclone method has surpassed the problems associated with the use of native mosses. In this sense, the utilization of cloned moss in a standardized way is a step ahead of this new policy.

1.5. Societal impact
Directive 2008/50/EC dictates to improve the information to the public concerning the ambient air quality but states are unable to collect this information. The MOSSCLONE technology will allow the appropriate data collection.
With citizens more aware of the situation and the risks involved, they are likely to get more involved when improving environmental health and deaths caused by air pollution will decrease. Besides, the exposure method is so easy that citizens may have pollution data from anywhere. They just have to buy the Mossphere, expose it for 6 weeks and sent back to analyze the preferred pollutants. Indoor monitoring may be an additional use by establishing a protocol of the exposure method for indoor use.

1.6. Environmental impact
The MOSSCONE technology can be used to map the deposition and dispersion of trace elements in Europe.
Importantly both the production and use of MOSSCLONE moss-bags are environmentally safe; its use do not need electricity or other power source, moss-bags are biodegradable and eco-friendly, the manufacturing consumes much less energy than currently used sample collectors and produces a minimal amount of waste generation (mainly organic and biodegradable).
Life Cycle Assessment of the method has defined the number of mosspheres that can be exposed while being more eco-friendly and representative than traditional methods.

2. Dissemination activities
The Mossclone project has been widely disseminated through different ways. The different categories are reported below:
2.1. Journal Publications
4 articles have been published on international, indexed and peer reviewed scientific journals. All the articles have been published with open access. Furthermore, there is still information from the project to be published and, at least, 4 more articles are estimated to be published with open access.
The last open access article has been published in mid may regarding Life Cycle Assessment of moss cultivation. The article has been published online and the participant portal does not recognize the related journal. The full and correct citation is shown below:
Carolina Alfonsín, Paula Pérez-López, Ana Isabel Rey-Asensio, Carlos Carballeira, Gumersindo Feijoo, Maria Teresa Moreira, 2015. Assessing the Environmental Performance of a New Biotechnological Sensor for Air Quality Based on Devitalised Moss Clone. Environmental Research, Engineering and Management, http://dx.doi.org/10.5755/j01.erem.71.1.10820.
2.2. Meetings
Both the kick-off meeting and the final meeting had a conference day opened to the public. Project stakeholders, authorities and the scientific community were invited to attend the meetings.
2.3. Conference Proceedings
Several posters and oral communications related to the Mossclone project have been presented by partners at more than 9 different conferences.
▪ 26th Convegno della Società Lichenologica Italiana (SLI) – Piacenza, Italia. 2-4th October 2013.
▪ Kyoto University Symposiums – Kyoto, Japan. 13-14th December 2013.
▪ 31st Convegno Nazionale Società Italiana di Chimica Agraria (SICA) – Napoli, Italia. 16-17th September 2013.
▪ 34th International Symposium on Halogenated Persistent Organic Pollutants – Madrid, Spain. 31st August- 5th September 2015.
▪ SECyTA 2013 - XIII Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines – Tenerife, Spain. 8-11th October 2013.
▪ III CICLO DE CIENCIA COMPARTIDA DE LA FACULTAD DE CIENCIAS DEL MAR – Las Palmas, Spain. 10th June 2014.
▪ Californian Goldschmidt – Sacramento, USA. 8-13th June 2014.
▪ XVII Seminario ibérico de química marina – Gran Canaria, Spain. 11-13th June 2014.
▪ 24th Earth science Meeting – Pau, France. 27-31st October 2014.

2.4. Invited talks
The scientific coordinator was invited to present the Mossclone project in more than 4 times, including public research facilities and higher education schools.
2.5. Website
A website for the promotion of MOSSCLONE and the exchange of information within the consortium is available at www.mossclone.eu. The project website was set up at the beginning of the project by Dr. Esther Schwarz-Weig (Conceptual Design) and has been continuously updated by Prof. Dr. Ralf Reski (representative of University of Freiburg, WP5 leader). The website includes two parts: one publically opened and one restricted (“intranet”) to the members of the Consortium.
2.6. Social Media
There is a Wikipedia article of the Mossclone project (English and German languages).
Mossclone Facebook and Twitter accounts were created to inform about Mossclone events and news.
-Facebook MOSSCLONE group has 68 members
-Twitter MOSSCLONE account has 65 followers
2.7. Media Briefings
National Spanish television news programs have informed about the Mossclone project 5 times. Other Galician tv programs have also reported 4 times the progress and meetings of the Mossclone project.
The Spanish national radio (RNE) also interviewed the Mossclone scientific coordinator and the Biovia representative.
2.8. Newspapers
The Mossclone project appeared at least 18 times in national and regional newspapers from Spain, Italy and Germany.
Moreover, the MOSSCLONE project has appeared in more than 25 digital newspapers and websites during project development.
2.9. Newsletters
Newsletters were published every 6 months in the Mossclone website (April and October). A total of 6 Mossclone newsletters have been published. Newsletters were uploaded and publically available on Mossclone website.
2.10. Documentaries
The basics from Mossclone project have been described in a documentary from Euronews channel. The program was called “Biotechnology to fight air pollution” and was part of the documentary series called “Futuris”. The documentary has been dubbed into 14 European languages. The link of the film is attached below:
http://www.euronews.com/2013/06/03/biotechnology-to-fight-air-pollution/
2.11. Project Dissemination Products
Several customized products were created during project development; posters, flyers, brochures, mousepads, pens, banners, USB memories, notebooks...
Moreover, 3 Mossclone videos have been created about:
- Description of moss samples processing
- Mossclone project development
- Mossphere for commercial usage
2.12. Sampling
Samples were exposed together with a banner in order to disseminate the project physically in the places where the experiment took place (see picture on the right).

3. Exploitation of results
A patent application including the whole exploitable foreground from the Mossclone project has been submitted at the end of the project. This foreground includes information regarding physical-chemical properties of the moss clone, the exposure method, analysis procedure of contaminants from moss samples, the cultivation method and the benefits of the moss clone method regarding traditional air monitoring techniques.
The acceptance or rejection of the patent application will be rapidly communicated to the European Commision when receive it.

List of Websites:
Web presence was a central element in the dissemination activities of the MOSSCLONE Project. The actual website is available at: www.mossclone.eu.

The content of the website has been managed by ALU-FR (University of Freiburg) in collaboration with MOSSCLONE project manager:
- Web designer: Esther Schwarz-Weig
- Web master: Ralf Reski (ralf.reski@biologie.uni-freiburg.de)
- Project manager: Carlos Brais Carballeira Braña (carlosbrais.carballeira@gmail.com)