Short-term monitoring of Arctic trace metal contamination based on Cetrariella delisei bioindicator in Svalbard

This study focuses on short-term monitoring of trace metals in the Svalbard archipelago. Short-term studies using lichen bioindicators are important because temporary changes in lichen trace metal levels are mainly dependent on air pollutants. Here, we investigated temporal and spatial diﬀerences in the content of trace metals such as Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, and Zn measured in the lichen thalli of Cetrariella delisei . The temporal aspect was studied in the marine plain of Calypsostranda between 1988 and 2016 and that of Hornsundneset between 1985 and 2008. The spatial aspect was studied between Hornsundneset in 1985 and Calypsostranda in 1988 as well as between Hornsundneset in 2008 and Calypsostranda in 2016. The results revealed an increase in the concentration of Cr, Mn, Ni, and Co for both the aspects, while a decrease in the contents of Cu, Cd, and Mo was observed. Pb content varied, as Pb level increased with time in Hornsundneset but decreased in Calypsostranda. The Zn content showed no signiﬁcant changes in both temporal and spatial aspects.

Climate changes observed in the Arctic region during last 30 years have significantly affected the tundra vegetation in Svalbard archipelago [14,15]. ese changes coupled with increasing herbivore pressures have led to a noticeable decline in the number of species of terricolous, fruticose lichens such as those from the genera Cladonia and Cetraria s. l. [14,16]. In the past, these genera had broad geographical ranges and were commonly present throughout the archipelago. As these genera have been the major components of the high Arctic tundra vegetation, they were frequently used as trace metal bioindicators [2,8,[17][18][19][20]. e studies involving lichens as biological indicators of trace metal contamination have mainly been conducted in the regions of Bellsund [21][22][23] and Hornsund [7,13,24,25]. e data of these reports have been used for comparative studies that are necessary to achieve the objectives of an AMAP ( e Arctic Monitoring Assessment Program) initiative [1]. One of the assumptions of this program is the assessment of temporal trace metal trend that provide essential information for decision makers connected with science-based policy decision on contaminants in the Arctic environment [1]. Short-term data (i.e., <30 years) are developed on the basis of the bioindicator studies (including lichens) and cover information from previous 1-3 decades. ese data illustrate how trace metal contents have changed in time and indicate their trends in the future. Short-term studies that use lichen bioindicators are also important because the changes in the levels of trace metals in lichens are not subject to strong fluctuations, as in the case of measurements of air contamination [26].
At present, the lack of the availability of the lichen material for comparative research poses a problem. Species of macrolichens that form large surface thalli, such as Flavocetraria cucullata (Bellardi) Kärnefelt & A. ell, F. nivalis (L.) Kärnefelt & A. ell, Cladonia mitis Sandst., and amnolia vermicularis (Sw.) Schaer., collected in the 1980s and 1990s on the coastal terrains of NW Wedel Jarlsberg Land and NW Sørkapp Land, now failed to occur in these regions owing to the heavy grazing activity of reindeer [13,14,16]. erefore, it is very difficult to repeat previous studies conducted on these species and compare the results with the original data.
Until now, the only short-term study for the period of 8 years has been performed on Calypsostranda plain [23] in the NW Wedel Jarlsberg Land; however, the results were not compared based on individual years but only averaged to have a better representation of the whole region. us, the short-term data sets from this part of the Arctic region are very poor. Within the monitoring survey, nine trace metals (Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, and Zn) were measured in the thalli of Cetrariella delisei (Bory ex Schaer.) Kärnefelt & A. ell. Here, we aim to investigate the spatial and temporal differences in the contents of these trace metals. e temporal aspect was studied for the marine plain of Calypsostranda between 1988 and 2016 as well as for the marine plain of Hornsundneset between 1985 and 2008. e spatial aspect was studied between Hornsundneset in 1985 and Calypsostranda in 1988 as well as between Hornsundneset in 2008 and Calypsostranda in 2016. e hypothesis set was as follows: the content of the studied trace metals in lichen thalli collected in Hornsundneset in 2008 and Calypsostranda in 2016 is lower than that in the samples collected in Hornsundneset in 1985 and in Calypsostranda in 1988.

Material and methods
Herbal material collection e study included the herbarium specimens of C. delisei lichen that were collected in Calypsostranda marine plains (NW Wedel Jarlsberg Land, Spitsbergen) and Hornsundneset marine plains (NW Sørkapp Land, Spitsbergen) by various researchers ( Fig. 1 and Tab. 1). Calypsostranda area shows rocks of tylloid in alternation with other rocks from sedimentary to low-grade metamorphic type, while Hornsundneset marine plain comprises rocks of sandstone, siltstone, and shale. For laboratory analysis, minimal quantities of lichen thalli were obtained from herbarium specimens that were stored in lichenological envelopes in the Herbarium of the Institute of Botany in the Jagiellonian University (KRA).

Laboratory analyses
Two replicates of 3 g from each lichen sample were homogenized and dried at 105°C, followed by mineralization and extraction in aqua regia for 16 h, according to a previously described method [27]. A er extraction, the samples were digested at 130°C for 2 h, filtered, and mixed in 0.5 mol nitric acid (HNO 3 ) in 100-mL flasks. e levels of Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, and Zn in each sample were detected in four replications with the Perkin Elmer Optima 7300 DV optical emission spectrometer using the inductively coupled plasma-optical emission. e plasma gas flow rate was 15 dm 3 min −1 , the external gas flow rate was 0.2 dm 3 min −1 , and the nebulizing gas flow rate was 0.6 dm 3 min −1 . Calibration was carried out using a certified reference material ERM-CD 281.

Statistical analyses
Levene's test was performed to assess the equality of variances and Shapiro-Wilk test was applied to assess normality. Wilcoxon test was used to investigate the differences in element contents in C. delisei thalli in temporal aspect between specimens collected in 1985 and 2008 in Hornsundneset as well as between those collected in 1988 and 2016 in Calypsostranda. Mann-Whitney U test was applied to test the spatial differences in element contents measured in lichen thalli collected in 2008 in Hornsundneset and 2016 in Calypsostranda as well as to analyze the difference between the data from 1985 and 1988. Statistical analyses were carried out using Statistica 10 so ware [28].

Results
Nine trace metals (Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, and Zn) were investigated in terms of their contents in the thalli of C. delisei collected in Calypsostranda in 1988 and 2016 as well as in Hornsundneset in 1985 and 2008 (Fig. 2). e differences in the temporal changes in the trace metal contents were significant between Hornsundneset in 1985 and 2008 for all the studied elements (Tab. 2). For Calypsostranda in 1988 and 2016, the differences in the changes in the element content were significant for the following trace metals: Co, Cr, Cu, Mn, Ni, and Pb (Tab. 2). However, the differences were not significant for the change in the content of Cd, Mo, and Zn between Calypsostranda in 1988 and 2016 (Tab. 2). e differences in spatial changes in the trace metal contents measured in lichen thalli were not confirmed for all the studied elements (Tab. 3). e differences were significant between Hornsundneset in 1985 and Calypsostranda in 1988 for following trace metals: Cd, Co, Cr, Mn, Ni, and Pb. e content of Co, Cr, Mn, and Pb showed significant variation between Hornsundneset in 2008 and Calypsostranda in 2016 (Tab. 3).

Discussion
Studies concerning short-term trends in trace metal contents measured in lichens thalli have not been conducted in Svalbard. is is probably owing to the lack of historical herbal materials that may be used for comparative research. e results show that the temporal changes in the trace metal contents in C. delisei thalli were not identical for all elements (Fig. 2). For the four trace elements Co, Cr, Mn, and Ni, an increase in the concentration was observed for both the studied areas (Fig. 2). ree elements Cd, Cu, and Mo showed a decrease in their concentrations (Fig. 2); however, the difference was significant only for Cu (Tab. 2). For Pb, the trends observed in both the areas were different; Pb concentration increased in Hornsundneset but decreased in Calypsostranda (Fig. 2). Zn contents in both temporal and spatial aspects showed no significant changes (Fig. 2). e hypothesis set herein was not confirmed by the observed results ( Fig. 2  caused the decrease in the levels of some elements with the simultaneous increase in the levels of other elements in a situation, wherein air pollution monitoring surveys clearly indicated decline in trace element concentrations [29]. e attempt to answer is a part of a long-year discussion based on numerous investigations that evaluate the problem of the accumulation of pollutants by lichens under various aspects. e most important ones are discussed below. Lichens are slow-growing and long-living symbiotic organisms that produce thalli, which lack roots or waxy cuticles and rely on an atmospheric input of mineral nutrients. ese features of lichens combined with their wide occurrence in Arctic areas make them good bioindicators of air pollution [30,31]. Despite these anatomic and morphological features, numerous studies have indicated the occurrence of a process of selective accumulation of individual elements within the thallus [32]. is phenomenon exists because the accumulation of trace elements occurs both at the intracellular and extracellular (in the spaces between the cells of lichen thallus) regions. In the thalli of lichens, the cations of trace elements bind to the extracellular anionic exchange sites that are located in the cell wall and plasma membrane surfaces [33,34]. Lichens as bioindicators have an advantage over other organisms, owing to their capacity to retain high amounts of contaminants in particulate forms in large intercellular spaces [35,36]. Studies have suggested that these cell wall-bound elements are readily exchangeable; therefore, extracellular amounts and proportions reflect the recent environmental input [37]. Furthermore, the accumulation of trace elements by extracellular secondary metabolites is a permanent phenomenon, which o en leads to morphological changes in the thallus at high concentrations of toxic elements [38]. Oxalates considered as one of the most effective extracellular mechanisms of heavy metal detoxification are widely distributed in lichens [39][40][41]. In the Arctic areas, such morphological changes fail to occur because of the low levels of contamination; however, the accumulation of the selected trace elements occurs in the intercell spaces in the form of permanent complexes. Total trace elements in the thallus of lichens are the most important fractions because the intracellular fraction accounts for only about 5% of the total content [42].
Trace element concentration in lichens is a dynamic process. Short-term research on the effects of excess metals showed that the lichens soaked into trace metal solutions accumulated elements quickly, in most cases within a few hours. In the case of Cu, maximum accumulation was observed a er 3 to 6 h [43]. Transplantation studies showed that most lichens respond to the changes in the atmospheric pollutants within a few months, while the residence time of many elements in lichen thalli is 2 to 5 years [44]. In transplantation studies of lichen thalli [44], the initial levels of trace metals were determined and the changes in the accumulation of elements in the thallus at a given time were monitored. us, the heavy metal content of lichens may increase as a function of time; however, the situation is much more complicated. In fact, the content of several trace elements in the transplanted lichens increased, while that of other elements decreased during the study period [36]. is observation may explain that the contents of these elements are, at least in part, controlled by physiological processes and turnover mechanisms [36].
In relation to Calypsostranda, Jóźwik [23] conducted numerous studies on trace metal levels in lichens between 1987 and 1995. In these studies, several species of lichens were used as bioindicators, including C. delisei. Studies in Calypsostranda area carried out in 1987 by Jóźwik [22] highlighted the differences between trace metal contents obtained in the currently analyzed samples of C. delisei from 1988. e samples from 1987 showed the following values: Mn, 5.82 ±0.42 mg kg −1 ; Cu, 3.58 ±0.25 mg kg −1 ; and Zn, 60.8 ±5.58 mg kg −1 [22]. ese values are four and two times lower than those reported in the present study (Fig. 2). However, the values of Cd (0.96 ±0.01 mg kg −1 ) and Pb (25.52 ±0.03 mg kg −1 ) were twice as high as those reported in the present study (Fig. 2). e method of mineralization of lichen samples is probably responsible for the observed differences. e lichen material was mineralized in a mixture of nitric acid (63%) and perchloric acid (60%) at 7:1 ratio [22], which is different compared to the mineralization process carried out using aqua regia, as performed in the laboratory analysis in the present study. ere is no universal dissolution method, but the results of the methodological study [45], wherein the plant material was thoroughly washed with water with subsequent ashing at 550°C and digestion with a mixture of HNO 3 :H 2 SO 4 (2:1), suggest that the method employed in the present study is suitable for the quantitative determination of trace elements in vegetation. e results [44] of the trace element contents in a standardized plant material show that the use of aqua regia in relation to Cd and Pb provides almost twice lower content than that obtained using a mixture of nitric acid (63%) and perchloric acid (60%).
Despite differences in the compared results from 1987 and 1988, the obtained results are similar except for Cu, as Cu content in 2016 was similar to that reported by Jóźwik [22].
In Hornsundneset in Sørkapp Land area, the first study on the evaluation of the level of contamination of trace metals in the lichen and bryophytes was carried out in 2013 [13]. In comparison to the obtained results, Ni, Cr, and Cu had similar values, while Mn and Zn had about five times higher values. Pb value was three times higher, while Cd concentration was ten times higher. e lichen materials collected in 2008 and used in the present study were obtained from the coast approximately 300 m away from the shoreline, while the material used for the research in 2013 [13] was collected from the slopes of the Hohenlohe ellet mountain at a distance of about 1.4 km from shoreline. e impact of the marine aerosol on lichens growing near the shoreline may have affected the levels of trace metals, mainly Cd and Pb [11]. Similar impact of marine aerosols on lichens has been reported while evaluating the accumulation of trace metals in a research conducted in the Kaffiøyra plain [11].
Almost all of the studied elements, except Mo and Zn, showed higher concentrations in Calypsostranda region in 1988 than in Hornsundneset region in 1985 as well as between these regions in 2016 and 2008 (Fig. 2). is observation may be associated with the influence of mining activity in the northwestern part of Wedel Jarlsberg Land [46] before the establishment of the Sør-Spitsbergen National Park. us, in this area, the source of trace metals accumulated in lichens seems to be not only the long-distance transport of pollutants with air masses but also the human activities. roughout Sørkapp Land, hunting was the only form of human activity before the establishment of Sør-Spitsbergen National Park. As a result, the only source of trace metals in lichen thalli is the long-distance transport of pollutants and natural geological background [13].
With regard to the other regions of the Arctic, our research shows that the accumulation of trace metals in a particular year in lichen thalli is not the same for all elements. is is important during the comparison of the data on the measurements of the trace metal contamination from air. For instance, short-term studies conducted at meteorological station Alert, Ellesmere Island station in Canadian Arctic [26], showed decreasing trends of concentration for Cu, Mn, Pb, and Zn in the years 1980-1995. While other Zeppelin Mountain station close to Ny-Ålesund, Svalbard [29], had not recorded any significant temporary trends in concentrations of Pb, Cd, Cu, Zn, Cr, Ni, Co, and Mn from 1994-2002. e results in this study do not correlate with our values of the trace metal recorded in lichen thalli, as the values of the four elements increased and similar trends were reported for only one element (Fig. 2).
In the Arctic region, the only temporal data available for lichens were from Nuuk (Greenland) where the samples of F. nivalis were collected in 1994 and 1999 [1,8]. e present study is the first report that analyzed short-term level of trace metals using lichens as bioindicators in Svalbard. Reducing pollutant emission contributes to the improvement in the conditions in the Arctic region. e monitoring of trace metal pollutants in air is very important; however, the response of biotic elements to the changes in the pollutants may only be assessed using the tried and tested bioindicators. Lichens, including, C. delisei, are good sources for the implementation of planned shortterm pollution measurements in the tundra region because of its widespread nature. Moreover, lichens are easy to identify and the thalli of this species are abundant; lichens form a significant element of the tundra region as the beginning of the food chain in the Arctic ecosystems. However, no information on the life expectancy in natural conditions is available for these species as well as for other fruticose species such as F. nivalis or Cladonia mitis. Correlation of life expectancy with trace element levels in the fruticose lichen may allow planning of monitoring studies while taking into account the actual rate of accumulation per unit time.