Lichen as biomonitor of atmospheric elemental composition from Potter Peninsula, 25 de Mayo (King George) Island, Antarctica

Lichens are powerful biomonitor of airborne pollution around point sources or long range transport because they are perennial allowing bioindication at long period. The element concentrations in foliose and fruticose lichen species from Potter Peninsula located in 25 de Mayo (King George) Island is reported. The coeffi cient of the variation for most of the elements was up to 50% except for as and Br, K and Se. The Principal Component Analysis showed differences among sampling sites according to human activities respect to the special protected areas. Aluminium, Cr, Hg, Pb and Se concentrations are linked local waste burning, global inputs, and the melt-water processes, while Br and Se were associated with marine biogenic cycle. This information could be a valuable tool for future atmospheric studies. Research Article Lichen as biomonitor of atmospheric elemental composition from Potter Peninsula, 25 de Mayo (King George) Island, Antarctica Mariana S Rivera1, Soledad Perez Catan2, Carla Di Fonzo3, Laura Dopchiz3, Maria A Arribere2, Martin Ansaldo3, Maria I Messuti4 and Debora F Bubach2* 1National University of Comahue, Quintral 1250, (8400) S.C. of Bariloche, Río Negro, Argentina 2Laboratory for Neutron Activation, Bariloche Atomic Center, National Atomic Energy Commission, Av. Bustillo km 9.5, (8400) S.C. of Bariloche, Río Negro, Argentina 3Ecophysiology and Ecotoxicology Laboratory, Argentine Antarctic Institute, Campus Migueletes, (1650) San Martín, Buenos Aires, Argentina 4Institute of Research on Biodiversity and the Environment, National University of Comahue, National Council of Scientifi c and Technical Research, Bariloche University Regional Center, Quintral 1250, (8400) S.C. of Bariloche, Río Negro, Argentina Received: 18 July, 2018 Accepted: 28 July, 2018 Published: 31 July, 2018 *Corresponding author: Débora F Bubach, Laboratory for Neutron Activation, Bariloche Atomic Center, National Atomic Energy Commission, Av Bustillo km 9.5, (8400) SC of Bariloche, Río Negro, Argentina, E-mail:


Introduction
Lichenized fungi (lichens) are well known as biomonitor organisms used to evaluate the air quality. The lack of a wax cuticle and stomata allows to nutrients and contaminants to be absorbed over the whole surface. The absorbed elements can be taken from the dry and wet deposition and by short and longrange transport [1][2][3]. Likewise, they can extract elements from the melt-water such as Al, Co, Cr, Pb, Mn, Ni and REE especially for Antarctic ecosystems [4].
The lichens reported data are mainly about global pollutants such as Pb, Cd, Zn, Cu, Cr and Hg [5][6][7]. The use of new materials of technologic application (nanoparticles), plus the increment of industrial and population development, and the effects of global warming could improve the transport, deposition and availability of pollutants. On the other hand, the increase of global temperature is heterogeneous and it occurs more rapidly in Antarctic and Arctic Continents [5]. Actually, it will need to extend the knowledge about the background levels of more elements as control tools due to those are being released to the environment [8]. For this reason, to expand the knowledge about the elements present in Polar Regions represents a safeguard for the future.
The Antarctic Protocol provided strict environmental management and protection guidelines, and established the obligation to clean-up abandoned work sites. Also, establishes principles for planning and conducting of all Antarctic activities. However, the local impacts due to the increasing of human presence seem inevitable [1]. In the 2008 Antarctic Treaty Consultative Meeting, some of these areas have been designated as Antarctic Specially Protected Areas (ASPA; https://www.ats.aq/e/ep.htm) with the purpose to preserve these environments due to their high sensitivity to disturbances. Element determination, concentration levels and potential toxicity, in key species are very important in order to understand and to elucidate the impact on the Antarctic terrestrial ecosystem.
Species of the genus Usnea has a wide distribution around the world including Antarctica and they have frequently used as bioindicator of presence of elements and compounds [1].
The element contents in lichens from Antarctica are widely reported in the literature, in particular several works reported the elemental composition in lichens from 25 de Mayo (King George) Island, South Shetland Islands, Antarctica [2,9,10].
However, the information about more elements and their concentrations in Antarctic lichens is still insuffi cient [11].
During three Antarctic summer campaigns, lichens sampling in Potter Peninsula located in 25 de Mayo (King George) Island were made with the aim to evaluate the effects of human presence in the area and the coastline ecosystems.
In the present work the concentration of 27 elements in lichens of the third last campaign are reported in order to identify the elements associated with the anthropic source or biogeochemical process. Those results were compared with our previous data.

Study area
The Potter Peninsula is placed at the southernmost end snowfall and scarce rainfall in summer; and strong winds predominant from the southwest sector [13]. The development of Antarctic terrestrial biotic communities is mainly limited by the freshwater availability, low temperatures and limited photoperiod [1]. Therefore, few groups of organisms can colonize or survive in this area; most of them are lichens and bryophytes that grow on the ice-free coastal areas (rocks and soil) during summer [14]. The ASPA-132, protected area is characterized as a wilderness representative area with high biological diversity and richness, which includes penguin colonies, elephant seals and sea lions, and skuas and petrels in the elevated zone, as well as dominant lichens in rocky formations of the highest sites and close to the beach ( Figure 1).

Lichen identifi cation
The study was based on the fresh material deposited in Centro Atómico Bariloche collected by the authors, and on desiccated selected material from BCRU herbarium to compare.
Specimens were examined and identifi ed using a stereo dissecting microscope (Olympus SZ30) and a light microscope (Leitz Laborlux 11). Anatomical features were studied on handcut sections mounted in water and in lactophenol cotton blue; 10% potassium hydroxide (10% KOH) or 1% Lugol´s Iodine solution directly and after a KOH pre-treatment. All measurements were made in water [15,16]. Secondary lichens compounds were identifi ed by High Performance Thin-Layer Chromatography (HPTLC) [17].
Three gamma ray spectra, with different decay times, were collected using an intrinsic HPGe detector 30% relative effi ciency and a 4096 channel analyser, whereas the spectra were analysed by using the GAMANAL routine included in Precision of analysis was estimated by the coeffi cient of variation of four replicates and was found to be within 10% for all elements. The limit of detection to the equipment was 5 μg/L and the LQ was 0.0125 μg/g.

Data analysis
The statistical analysis was performed with XLSTAT program (Copyright 1995-2009, Addinsoft). When the concentration of an element was below the detection limit, one third of this value was used for statistical analysis and descriptive purposes. Data were analysed using a multivariate statistical method. Principal Component Analysis (PCA) using the elements contents as quantitative variables was applied.
The considered signifi cance level in statistical tests was  ≤0.05.
Agglomerative Hierarchical Clustering (AHC) based on Spearman correlation coeffi cient over the element concentrations in the lichens was used in order to evaluate comparatively the elemental composition in each sites (S1-S8) with respect to the lichen habitus (e.g. foliose vs. fruticose).
The dendrogram was based on Similitude Index with average link method. The concentration of element (X) and samarium (SM) with subscript "liq" designates the lichen sample and "sub" for the soil content in sample of the same sites [19]. The EF(x) values greater than 1 indicates that the element in the lichen is enriched compared to the substrate. In this work, an element was considered enriched when EF(x) values were greater than or equal to [5].
Pollution Load Index (PLI) was used to assess the environmental quality of the area, and it represent one specifi c site. The formula used by [20], takes into account the load of all elements given by:

Species evaluation
Specimens collected in Potter Peninsula are shown on Table   1 Table 2, and the PCA results in Figure 3. The EF(x) values of non-lithophile elements by sampling sites are presented on Table 2, in general, the S6, S7 and S8 were the sites with the highest EF values for Br, Ca and Hg. However, Al and Se were enriched in all sites. In adition, Br presented high EF in S2 and S3, and it was observed in S1 by Pb and Hg.
Polution Load Index (PLI): The site used as control for PLI calculus was S7. The choice was due to the fact that this site presented the lowest concentrations for the most elements (Supplementary Data, Table 2). Figure 4 shows the PLI values, being S2 the more deteriorated site with PLI to 3.47, followed by S8 (PLI: 2.26) that also presented a high level of deterioration; the lowest PLI (1.07) was for S6 site.

Discussion
Lichens accumulate elements, some of them environmental pollutants, for a long period, in excess of 100 years due to their longevity [22]. The relation between the growth form and the accumulation capacity has been reported as a factor that  affects the element accumulation patterns in lichens [23,24]. The results in Supplementary Data, Table 2 showed for the mostly elements, e.g.: Sb, As, Ba, Cs, Co, Hf, Fe, La, Rb, Sc and Sm, major concentrations in foliose than fruticose form, while for Br, Ca, Hg and Ag were similar in both habitus taking account the average, standard deviations and median of the concentrations. Our results are agree with some authors whose suggested that foliose form has the highest exposed surface to the atmosphere and this related to higher ability to incorporate elements [25].
The dendrogram Figure 2 showed high similarity index (0.964) for homogenous grouping to the family Parmeliaceae with respect to the other clades belong to the family Physciaceae. This may be a coincidence. The criteria on the lichen family taxa somewhat ambiguous and depends on the authors. Genus Usnea and Himantormia are classifi ed within the Parmelaceae family which is very diverse and including two foliose and fruticose habitus (Thgorsten et al. 2012). In other hand, genus Usnea is also classifi ed in the Usneasea family excluding the genus Himantormia [26].
The dendrogram nest the lichens of for their habitus in spite of the fruticose S. globosus is an exception, which is grouped together with the foliose P. muscigena; unlike the other fruticose species, S. globosus, was found in association with bryophytes (mostly mosses). The growing condition in high humid microenvironments could make to the elements more available for the lichens [27]. Environmental changes have considerable infl uences on physico-chemical process such us pH, redox potential or oxygen diffusion rates [27]. The nutrients and elements can be leached and extracted from the colonized rock, roughened by molten water that fl ows downward and can seep into the cushions of lichens and mosses. Those processes could explain the high concentration of some elements in S. globosus and the ensemble showed in Figure 2 with fruticose lichens. In addition [28], found in mosses samples from dry and barren Antarctic terrestrial ecosystems, that raw concentrations of elements often refl ect the biogeochemical nature of soils and rocks rather than atmospheric input, generally Al, Fe, Cr, and other lithophile elements.
The PCA of the Usnea sp., showed clearly a distinction of sampling sites, the sites near to Carlini Station (S1 to S5) had a major contribution of elements, which could related to the human activities like was shown in previous studies at the 25 de Mayo (King George) Island [1,2]. The reported results in this work together with our data from the two previous sampling campaigns showed the same trends about the element effects on antrophic and ASPA area [10]. A great variation in the element concentrations was observed in all champagnes, the coeffi cient of variation lower than 50% was found for as and Br, K and Se (Supplementary Data, Table 2).
The ACP-graphic in Figure 3 includes Al and Pb contents producing the spread of sampling sites in slightly different way to presented by [10], where S2 is in other different quadrant that S1, S4 and S5.
The burning of waste as plastics, paper, batteries, and fuel oils, is most important activity that releases ashes with Hermanos hill, emphasize the relevance of leaching process followed by thaw waters [29]. These are in agreement with [30], whose inform several heavy metals concentrations as Pb, Cd, Fe and Cr in surface sediments from Potter Cove.

This authors suggest that most of the metals found in Potter
Cove constitute a redistribution of autochthonous materials within the ecosystem, and its can be considered to be present at natural background levels in surface sediments. Those informed processes in the literature, have not doubt that they contributed to dispersing elements in the study areas.
The site S4, is a place close to Tres Hermanos hill, was used like garbage dump until 1998 when the Madrid Protocol (1991) came into operation and remediation works were carried out [31].
The S2 samples correspond to the heliport area and they had a major lithophile element contributions as Ba, Hf, Fe, La, Sm, Sc and Th, which may be associated with the resuspensions of the surrounding sediments; Pb and, an important Cr contribution observed in S2 could also came from the emplacement of the fuel cisterns. This site and S4, moreover could be infl uenced by the water from Tres Hermanos hill, while S1 and S5 are affected by the melt water come from the Icefi eld [29,30]. The sites far away from the human settlement (S6, S7 and S8) were separated from those close to Carlini Station (S1 to S5) by the greatest contribution of Br, biological elements such as Se, Zn, K and pollutants as Hg, Ag and as. These sampling sites are included in the ASPA-132 where there are several marine mammal settlements and seabird colonies of penguins, skuas and seagulls. The bioconcentration of pollutants elements and detoxifi cation process in mammals and sea birds from Arctic and Antarctic area have been known base on excrement, guano and feathers analysis among others [32][33][34][35][36]. These authors proposed process sea-land bio-transport of chemical pollutants as As, Hg, Se and Ag.
The PCA results were in agreement to the EF (Table 2)  campaigns which can be able to biogenic marine cycles, while the other elements could be indicate anthropogenic impact [3,10,18,37]. Nevertheless, [4], found increased level of elements such as Al, Cr, Pb and other REEs, in water and glacier meltwater from 25 de Mayo (King George) Island. This represents a signifi cant impact on the biogeochemistry of coastal seawater in Antarctica and it could explain the high Al and Se EF values in all studied sites and the Pb EF in S7. Moreover, biomethylation is known as an important chemical process in the ocean, which can lead to volatile compounds of heavy metals, likes Cd, Hg, Pb, Ti and halogens as Br. Some results reported by [38], demonstrate that the bacteria and microalgae from the polar ocean are potential sources for the production of methylated heavy metals. These take place principally in the pack-ice section, whereas in the polar ocean under the closed ice sheet and the shore of the Antarctic Peninsula, methylated heavy metals diminish. Despite of the stability of the compounds in seawater is different, there are no doubts that they play an important role in the biogeochemical cycle of this regions like suggested by high EF values in pristine sites (S6 to S8).
The PLI values could be considered as contradictory to the EF. This is because S2 was the most deteriorate site, although in general, the EF values for this site were low. The PLI is a standardized method for the comparison of different geographical areas which take account all the elements, and indicates the background level when PLI is equal to 1 [39]. When it value increases, this indicates different pollution degree in the environment, whereas the EF values is particular for each element and considered soil. These explain the differences interpretations about both indexes in each area, in particular, PLI in S1and S2, and EF in S1, due to the high concentrations in the soil, for example, Cr and Pb by accidental fuel oil spills (Supplementary Data, Table 3).
The elemental contents in S8 showed a homogeneous contribution including As, Br, Ag and biological elements (Supplementary Data, Table 2). In this site, the important biological activities related with animal colonies and biogeochemical cycle, were the reasons, which justifi ed the designation as second deteriorated area by the high PLI.

Conclusion
Based on three sampling campaigns, the area close to the Carlini Station (S1 to S5) is enrichment of different elements respect to the sites far away from it, which includes in the ASPA-132 (S6 to S8). The most important remarks are: The presence of the pollutants elements related to anthropogenic sources as the local waste burning (Al, Sb, Cr, Pb, Hg, and Se), soil resuspensions (e.g.: lithofi le elements) and, fuel spills (Pb and Cr) also, the global inputs (Cr, Pb and Hg).
The natural pollutants and the process like soil leaching and melt water from glacier (Al, Cr and Pb) and biogeochemical process (Br, Se, Hg, Zn, Fe, Tl) are identifi ed as an infl uence in the elements contents of the Antarctic lichens. Furthermore, the results of our work support the idea that the marine biogenic cycle and sea-land bio-transport are the main processes that would affect the coastlines environments.