Bioaccumulation of trace elements in lichens exposed to geothermal and volcanic activity from copahue-caviahue volcanic complex, patagonia, Argentina

ASTM: American Society of Testing Materials; BCRU: Regional Center Bariloche Herbarium from the National University of Comahue; Río Negro; Argentina; CVCC: CopahueCaviahue Volcanic Complex; EF: Enrichment Factor; GT: Geological Tracer; IAEA: International Atomic Energy Agency; INAA: Instrumental Neutron Activation Analysis; m a.s.l.: meters above sea level; P: Lichens of Protousnea Genus; PCA: Principal Component Analysis; PC1: Principal Component1; PC2: Principal Component2; PM: Particulate Material; QC: Quality Control; REEs: Rare Earth Elements; U: Lichens of Usnea Genus; Sampling Sites; A: Achacosa pond; AR: Las Lecheras Stream; CA: Caviahue Village; CC: Caviahue Camping; CO: Copahue Village; E: Escondida Waterfall; H: Huelcupén Lake; M: Las Mellizas Pond; R26: Site Near to the Airfi eld; T: Trolope Pond

and about 80 are active today [3]. The aerosols emitted by volcanoes are considerably enriched in toxic components such as sulphur dioxide (SO 2 ), hydrogen sulphide (H 2 S), hydrochloric acid (HCl), hydrofl uoric acid (HF) and other minor components including trace elements as alkali metals, alkali earths, and transition metals [4]. It has been shown that amounts of Cd, Hg, Se, Cu and Zn are comparable to anthropogenic sources in the Mediterranean area [5]. Environmental effects on local, regional and global scales can be signifi cant depending on volcano characteristics and the processes that determine the magnitude of volcanic eruptions. The overall impact is a consequence of chemical and physical factors [4].
Control and monitor of air quality it is very important, at regional and/or global scales for adopting actions to protect the ecosystem and the people. The environmental quality can be evaluated by means of bioindicators such as lichens [6,7]. Likewise, in several cases they are used as quantitative tool owing to their ability to accumulate elements, some of them in relation to the environmental levels [8,9]. Qualitative determinations can be employed to determine the degree of environmental alteration. Contaminants can cause a decrease in the diversity and abundance of the lichen species and, in extreme cases; they can disappear from the impacted areas.
Although the time trend of contaminants can be faithfully recorded by the artifi cial samplers or one-line measurement equipment, the lichens indicate the bioavailable fraction of the contaminant. Active volcanoes emit SO 2 in the order of tons and the S concentrations reported in lichen for those volcanic zones are usually 0.5-1.5 mg/g [10][11][12].
In the Southern Volcanic Zone (SVZ) in South America, the direct impact of Puyehue volcano eruption, in 2011 was studied using lichens by Bubach et al. [4,13] and by Nelson and Wheeler [14] in areas up to 100 km from the crater. Also Conti et al. [15] evaluated the effects of the impact at 1600 km approximately from the volcano, in Tierra del Fuego, Argentina.
The volcanoes in the Andes Mountains play an important role in global climate change and the composition of the atmosphere in the Southern Hemisphere [3]. There are approximately more than 200 potentially active volcanoes along the Andes Mountains of South America [16]. Copahue is an active stratovolcano located in central Chile, in the Central part of the SVZ, at the Argentina borderline, integrating the Copahue-Caviahue Volcanic Complex (CVCC). It shows an extensive record of recurrent explosive eruptions since 1750 to the present [17], which affected the Copahue zone, a natural protected area with aboriginal settlements and tourist recreation.
Previous studies of the elemental composition in lichens after the Puyehue volcano eruption in 2011 showed some element concentrations were related to the volcano distance and the preponderant plumes [4]. In addition, the geothermal activity is another natural source of toxic compounds and elements as sulfhydryl acid (H 2 S), As, Br, and Hg for the environments [12,18]. Based on these results it is expected to fi nd that the elemental compositions in lichens from CCVC will present differences in geothermal zones from distant areas in a gradient affected by predominant direction of the ash plumes.
The aim of this work is to check the infl uence of the distance to the volcano crater, preponderant plumes and thermal areas over the element concentrations accumulated in fruticose lichens in the surroundings to the CCVC.

Study area
The CCVC rises in a sector of the Northern Patagonian Andes, in the Neuquén province, Argentina (37º 47'-37º 55 'S and 70º 55'-71º 10' O). This sector is a plateau with elevations exceeding 2000 m a.s.l. and characterized by glacifl uvial valleys at the northeast sector (Puerta del Trolope) and a considerable amount of wetlands associated with water springs, pools of water and bubbling mud, and fumaroles [17]. The vegetation is dense in the adjacent area to the watershed, alternating with semi desert and steppe vegetation in the peripheral areas. The Araucaria araucana (araucaria or pehuén) forests on slopes and rocky outcrops below 1800 m a.s.l. are characteristics of this region [19].
The emissions of the recurrent explosive eruptions of Copahue volcano are swept eastbound by the dominant westerly winds, following the valley of Caviahue and Trolope.

Sampling and sample preparation
The sampling areas were chosen considering the plume directions of the most infl uential Copahue eruptions since 1992 until 2016 and also, the permanent gas emissions from the crater and hot springs ( Figure 1). All the sampling sites in the areas are located towards the east of the volcano, which are listed and described on Table 1.
Specimens of Usnea and Protousnea species were collected according to their presence and availability (random walk) in March 2017 (Table 1). Thalli of Usnea sp. were collected from rocks, approximately 50 cm above the ground, and Protousnea sp. were taken from Araucaria araucana at least to one meter above the ground. A surface soil sample was taken in each area where the lichens were collected.
The samples were collected and processed using latex gloves and a titanium knife, and stored in sterile polyethylene bags. A fraction of lichen specimens were separated for later identifi cation. The lichens for the elemental analysis were conditioned based on the IAEA biomonitoring of air pollution by Instrumental Neutron Activation Analysis (INAA) methodological recommendations [20,21]. The residues of substrates and other non-lichen materials were removed under dissecting stereo microscope at room temperature (22°C). Afterwards, the lichens were submerged for two seconds in ASTM grade 1 water three times and dried in a laminar fl ow hood. Composite sample from each site was done with fi ve to ten lichens thalli of similar size and characteristic. Afterwards, composite samples were thinly cut, homogenized to a fi ne powder, and aliquots (130 to 180 mg) were put in quartz ampoules for analysis.
Citation: Bubach  Surface soil samples, taken at 2 to 3 cm deep with plastic spoons were placed in plastic jars with screw caps. All the samples were kept frozen at -20°C until lyophilization. Dried soils were sieved with a 125 μm mesh and aliquots of 50 to 100 mg were weighed in plastic containers for the analysis.
The geographical location data, such as latitude, longitude,

Lichen identifi cation
The lichen specimens were identifi ed based on fresh selected material and on herborized specimens deposited in BCRU herbarium to compare. A standardized protocol for lichen studies was used to identify each specimen [22] and they were examined using dissecting stereo microscope (Olympus SZ30) and a light microscope (Leitz Laborlux 11). Anatomical features were studied on hand-cut sections mounted in water and in lactophenol cotton blue; 10% KOH or 1% Lugol´s Iodine solution and after a 10 % KOH pretreatment.

Elemental analysis
The elemental composition of samples was measured by (Ta) and Zinc (Zn). Analytical errors were computed as the propagation of the uncertainties associated with the nuclear parameters, the effi ciency of the gamma-ray detection system, the neutron fl ux determinations, and the area of the specifi c emission considered.
The lichen samples were analyzed together with the IAEA 336 Lichen Reference Material for analytical Quality Control (QC), showing good agreement with the recommended values; these results are reported on Table 1 and Table 2

Data analysis
The normality of the element concentrations in the lichens was tested applying the Kolmogorov-Smirnov Tests and it is shown on Table 3, Supplementary Material. Principal Component Analysis (PCA) was performed on the element concentrations and geographical parameters (distance, altitude, longitude) as variables using the Spearman rank correlation. The signifi cantly related variables are highlighted in bold.
The soil samples composition include lithophile elements, such as the REEs, which correlate linearly among them. For this reason, the elements from soil particulate entrapped in the lichen thalli were identifi ed by linear regressions with a lanthanide element used as a Geological Tracer (GT) based on the Pearson Test. Samarium is usually proposed as GT because it normally presents the lowest analytical uncertainty respect to other REE when samples are analyzed by INAA [13].          The EF values of the elements that were >1 at least in a single site are shown on Table 4. Bromine, Se, and S were the most enriched (EF up to 70), and Sb, As, Ca, K, Rb, and Zn were the less enriched (EF: <10).

Lichens of genus
We compared in a descriptive way the elemental concentrations measured in this work with the available data for both fruticose and foliose lichens from volcanic and geothermal active areas of the world. The data are shown on Table 5.      (Table 5). This is consistent with Tamburello et al. [29] who reported that Copahue volcano crater lake has the highest SO 2 concentrations in the world.

Areas close to Caviahue lake (As, Sb, Br, Cr and lithophile elements)
The correlation among As, Cr, Br, Fe and Sb elements grouped the lichens from CA and R26. Those elements, except As, could be associated with the PM in spite of absent relation with GT. Scandium is a transition metal with similar characteristics to lanthanide elements and the GT, which correlated with the volcano crater distance (p≤0.05, Table 2) and with Cr, Br, Fe and Sb. These correlations allow to link these elements with the volcanic ash plumes.
Copahue´s 2000 eruption was probably one of the substantial contributors to elemental composition in lichens from the CA, CC and R26 areas. The ashes deposited during the event were several centimeters more than the others areas. In addition, Delpino and Bermúdez [30] reported that the ashes were enriched in S, As, Ta, and Ti.
The main ash deposition site induced by atmospheric conditions (wind, rain and snow) and topography was located close to the Caviahue lake ( Figure 1 the lower concentration of toxic elements (Hg and S) could indicate that these lichens have been less damaged than those close to the volcano crater (M, CO and PA); Figure 2a and 2b. Although, K and Mg could come together with the PM, they are soluble; they do not maintain their proportions with those lanthanides and being essential elements are bioaccumulated by the lichens. Potassium concentrations in lichens have been observed to decrease subsequent to cell membrane damage following pollution by SO 2 and heavy metals [6]. The effect of SO 2 fumigation induced changes in chlorophyll fl uorescence, indicating photosynthesis inhibition [35].
The absence correlation of Se with the GT in the lichens and the high EF Se values in the lichens evidence a great contribution of this element in the gas form. Predominant plume direction in the 2012 and 2016 eruptions [28,36] may have had a great infl uence on Se accumulation in the lichens over these areas. This is consistent with Se content in pyroclastic material from the 2012 event and the high concentrations measured in leachates in ashes from 2016 eruption [36,37].

Conclusions
Lichen elemental composition refl ects the impact of the volcanic eruptions and the permanent geothermic activity according to what was expected. The concentrations of the elements in the lichens are mainly infl uenced by the crater volcano distance and the wind. This last factor plays the most important role in the dispersion of the Copahue volcanic products as ash plumes, degasing from hydrothermal vents and evaporation from the water bodies.
The Agrio Upper river is the greatest impacted site where lichens are totally absent. Likewise, the sites on the northern edge of the Caviahue lake could have an anthropic contribution overlap those volcanic products.
The concentrations of nontoxic elements and the higher of the biological elements (Ca, K, Se, Mg, Mn) in lichens from Hualcupén lake and Las Lecheras stream, the most distant areas from the volcano crater, allow to classify them as the less impacted areas.
Sulphur concentrations found in lichens from the Copahue-Caviahue Volcanic Complex, as well as those previously reported for Puyehue-Cordón-Caulle Complex, are two to three times higher than other volcanic areas of the world.
The results of this study strengthen the practice on the use of lichens for bioindicator/biomonitor studies in geothermal areas as natural laboratories.
The permanent Copahue-Caviahue Volcanic Complex activity provides a favorable scenario to continue investigating the response of lichens to these impacts.