Significance of moss pretreatments in active biomonitoring surveys

Abstract The present study examines the impact of pretreatment procedures on the metal concentrations in bags that are to be exposed. We examine Mn, Fe, Cu, Zn, Cd, and Pb amounts in Sphagnum fallax and Dicranum polysetum mosses using atomic absorption spectrometry. The concentration of Hg was also determined using a mercury analyzer. Two sample preparation ways were tested (with and without rinsing) and their influence was evaluated by determining the coefficient of variation (CV). Chlorophyll content was also determined in mosses collected from three habitats (deep woodland, forest road, and wood lot). The results indicate, that the concentration of elements deposited in mosses depends on the species and the habitat where they were collected (ANOVA, p < 0.001). Rinsing of mosses reduces the CV for Mn, Fe, Cu, and Zn and uniform the material prior to exposure (CV for the majority of metals <10%). Selected correlations were found for element concentrations with chlorophyll content. Photosynthetic activity of mosses decreased by about 80% during their one-month storage in the laboratory. Due to the varying concentration of metals in the collected samples, proper, and standardized preparation of mosses before exposure, they can be effectively used in active biomonitoring. NOVELTY STATEMENT Compared to other biomonitoring work the novel approach is the simultaneous study of two moss species, the analysis of three different habitats and the tie-in of accumulated trace elements by mosses and their vitality by measuring chlorophyll content and photosynthetic activity.


Introduction
Mosses, used as species in passive biomonitoring or as transplants (active biomonitoring: moss-bags), can accumulate airborne pollutants, representing a cost-effective useful tool for monitoring atmospheric deposition (Temple et al. 1981;Di Palma et al. 2016).This is in response to the high cost of standard analytical methods and the difficulty of conducting extensive monitoring over time and space (Ani ci c et al. 2009).The use of the moss bag technique can be particularly useful in all extreme environments (e.g., volcanic tops), where atmospheric pollution monitoring stations will not be able to work (Calabrese et al. 2015).However, to be routinely used by public authorities as a reliable tool for estimating air quality, a standardized protocol should be developed (Ares et al. 2015;S , tef anuț et al. 2019).The standardization of moss biomonitoring has been the main research focus of many studies (Ares et al. 2012;Fern andez et al. 2015;Aboal et al. 2017).This is especially important for the bag technique in active biomonitoring (Ares et al. 2014;Capozzi et al. 2016), for which new application perspectives are still being proposed (Sorrentino et al. 2021;Bertrim and Aherne 2023).The basis, however, is the correct collection and preparation of samples (Dołe Rgowska and Migaszewski 2015; Dołe R gowska 2016; Burrascano et al. 2021), which is an important part of the protocol for international moss surveys (ICP Vegetation 2020; Betsou et al. 2021;Menda s et al. 2021).In the case of sample preparation, the same method should always be used by different research groups throughout all biomonitoring surveys and studies (Aboal et al. 2008).The sampling location, such as the presence of canopy above moss sampling site, is also influential ( Ceburnis and Steinnes 2000;Meyer et al. 2015) as well as have been rinsed with distilled water (Fern andez et al. 2010).The latter is particularly important because the selection of the washing method depends on which moss mechanism for analyte accumulation is under investigation, for example, bioconcentration or surface adsorption of analytes (Spagnuolo et al. 2013).Short rinsing time (30 s) in double distilled water are tested to remove particles adhering to the moss surface (Fern andez et al. 2013).The method of preparation, dry/wet cleaning of samples in turn affects the results of metal concentrations in mosses (Dołe R gowska and Migaszewski 2019).The contribution of environmental stress is also significant (Roma nska 2020).The influence of factors such as desiccation (Slate et al. 2019), salinity (Gupta and Seth 2023), season (Vukovi c et al. 2016;Klavina et al. 2018) or light (Dziurowicz et al. 2023) can cause a decrease with photosynthetic activity what is an important parameter in biomonitoring studies (Capozzi et al. 2020) in the context of using the washing (Capozzi et al. 2018).
The method of storage and sample preparation are the most important steps in the entire analytical procedure (Dołe Rgowska and Migaszewski 2020).Studies conducted under controlled conditions indicate that washing (simulated washing by rain) does not always remove adsorbed metals, indicating permanent binding of the element to the moss surface (Sabovljevi c et al. 2018).This is also supported by other studies which indicate that not all material associated with the moss surface was removed by rinsing (Motyka et al. 2019).Nevertheless, this procedure is important because washed mosses show chemical homogeneity (Adamo et al. 2008) and element concentrations in mosses before exposure in active biomonitoring are reduced compared to unwashed mosses (Calabrese et al. 2015).This is also confirmed by Swisłowski et al. who compared different methods ( Swisłowski, Kosior, et al. 2021).Homogeneity of the moss provides better replicability of the results, in case of short-term moss bag exposure (Ani ci c Uro sevi c et al. 2022).
The aim of this study was to: (1) find out variation in the concentrations of selected heavy metals in mosses of two species: Sphagnum fallax and Dicranum polysetum, which were collected in three different habitats in terms of atmospheric pollution levels, (2) to find out the influence of pretreatment regarding rinsing to demonstrate qualitative differences in the preparation of samples prior to exposure under the active biomonitoring moss-bag technique, and (3) to examine the relationships between vital parameters of mosses collected from different sites and elemental concentrations in their gametophytes.

Moss sampling and treatments
Sphagnum fallax (Sp) and Dicranum polysetum (Dp) moss species were used in the research.S. fallax is medium-sized, green to mustard-brown, growing in carpets.Branch leaves are weakly to clearly in straight lines.Plants with stem leaves clearly pinched at the tip are S. fallax.Very common in a wide range of permanently damp or wet habitats, including nutrient-poor to intermediate fens, and pools and runnels on bogs.D. polysetum, on the other hand is a robust moss, forming open patches up to 15 cm tall.The approximately 7.5-10 mm long leaves are narrowly spearheadshaped with the upper ones erect and those below spreading and only occasionally somewhat curved.The stem is covered by dense, felt-like rhizoids.A lowland moss of the ground in coniferous woodland and plantations, but also occurs in healthy birch woodland and on mires (Atherton et al. 2010).The mosses were collected in accordance with national regulations (hand collection, no less than 75% of each sod was kept), which limit the collection of mosses to a few species (Minister of Environment 2014  (Gałuszka 2007;Rajfur et al. 2016;Szwed et al. 2020;Kozłowski and Strzy_ z 2021).That confirms only the validity of continuing holistic biomonitoring studies in the Holy Cross Mountains area from the last 25 years (Dołe R gowska et al. 2021).Both species of mosses were sampled at all locations.Samples were collected in three different habitats: in deep woodland [A] (51 7'19.61''N, 20 29'52.78"E), on a forest road [B] (51 8'15.17''N, 20 29'54.71"E)and in the wood lot in the suburbs of the city [C] (51 8'6.71''N, 20 29'36.47"E).Sampling was performed in accordance with ICP Vegetation guidelines (ICP Vegetation 2020).The selection of three different habitats was intended to test the initial deposition of metals in the mosses and the effect of rinsing efficiency on reducing elemental concentrations.Sampling and rinsing methods were consistent with previously established methodologies and conducted research ( Swisłowski, Kosior, et al. 2021).The study was extended by inclusion of additional moss species in order to demonstrate the legitimacy of the method's and its possible application throughout all active biomonitoring studies.

Methods
Mosses from habitats A-C were prepared by two ways: only removal of adhered materials/impurities (e.g., leaves, needles, and soil) without rinsinsing [unrinsed way-U] and rinsing of mosses in demineralized water [rinsed way-R] with conductivity of j ¼ 0.50-1.00lS/cm for 1 h ( Swisłowski, Kosior, et al. 2021).The mosses were taken at the same time, that is, on a sunny day with no precipitation occurring for a week.The mosses were taken before noon, after the morning dew had fallen.Only green part of the gametophyte, live, and active tissues was used in the study (Boquete et al. 2014).In the next step, after removing leaves, needles, or soil particles, samples of mosses were manually mixed up (homogenization).All samples were conditioned together and left in 12 L of demineralized water ( Swisłowski, Kosior, et al. 2021).The rinsed samples were laid out on paper and left to dry from the excess water absorbed during rinsing under laboratory conditions (Motyka et al. 2019).A total of 144 samples were analyzed (12 samples per site Â 3 habitats Â 2 species of mosses Â 2 treats).

Analytical determinations
Each moss sample with a mass of 1.000 ± 0.001 g dry mass (d.m.) was digested in a mixture of nitric acid and hydrogen peroxide in the 5:3 proportion using a microwave oven (Speedwave Four, Berghof, DE).Before mineralization, the moss samples were dried at room temperature until dry mass was obtained.Samples were not pulverized before digestion.The concentrations of six metals (Mn, Fe, Cu, Zn, Cd, and Pb) were determined by using an atomic absorption spectrometer AAS (iCE 3500, Thermo Scientific, USA).The calibration of the spectrometer was performed with a standard solution from ANALYTIKA Ltd. (CZ).The values of the highest concentrations of the models used for calibration (7.5 mg/dm 3 for Mn, 10 mg/dm 3 for Fe, 5 mg/dm 3 for Cu, Zn, and Pb) were approved as linear limits to signal dependence on concentration.The concentrations of metals were determined in solution after digestion and filtration into 25 cm 3 volumetric flasks.Information on quality control can be found in Tables S1 and S2 in the Supporting information (SI).The concentration of Hg in the undigested moss samples (0.04 g ± 0.001 g d.m.) was determined with AMA 254 mercury analyzer (Altec Ltd., CZ).Metals of anthropogenic origin, which are often found in polluted air, were analyzed here in this paper, as well as because they can be compared to studies performed by another authors (Shvetsova et al. 2019;Sergeeva et al. 2021;Mao et al. 2022).The chlorophyll content in mosses was measured using a Cary 3500 UV-vis Compact Peltier spectrophotometer (Agilent, USA).For analyses on the spectrophotometer, mosses (0.1 g) were divided into small pieces and ground in a porcelain mortar with 5 mL pure acetone.Centrifuge (MPW-351RH, MPW Med.Instruments, PL) was first cooled, and then samples were centrifuged in it (10 min, 10,000 RPM).Each measurement was made in five replicants.Chlorophyll a and chlorophyll b analytical standards (ChromaDex, USA, certified dye content >97%) were used in liquid form.Based on the obtained absorbance values, the chlorophyll contents (chlorophyll a and chlorophyll b) were calculated using the extinction coefficients and equation (Lichtenthaler and Wellburn 1983).Chlorophyll fluorescence of Photosystem II (PS II) was monitored using the modulated portable fluorometer (Opti-Sciences, USA).These measurements were taken in the forest during moss collection and one month after storage in the laboratory after chemical analyses.Current photochemical efficiency (yield) was measured under ambient light conditions ( Sraj Kr zi c and Gaber s cik 2005).

Data analysis
The results were interpreted based on the coefficient of variation (CV), which refers the value of ratio of standard deviation s (absolute differentiation of the feature distribution) to the mean value of xm: (Konieczka and Namie snik 2018).This is frequently and commonly used in analysis of biomonitoring research (De Agostini et al. 2020;Messager et al. 2021;Vergel et al. 2022).Statistica (ver.13.3), JASP 0.10.2, and Microsoft Excel 2016 softwares were used to process and present the data.Shapiro-Wilk's test was used to check data normality.Analysis of variance (ANOVA) statistics were used to test whether metal concentrations in moss samples differed significantly between collection habitats and between species in the study habitats, while Spearman correlation ratios were used to determine whether there were strong and significant associations between vital parameters of mosses for the elements analyzed.

Results
The concentrations of heavy metals accumulated by two moss species collected from the three different habitats are shown in Table 1.
The mean concentrations shown in Table 1 indicate the differences between habitats in terms of their heavy metal contamination, and also indicate the variation in the accumulation of elements by two selected moss species.The ANOVA analysis applied for the measurements of two moss species and three locations confirmed significant differences at the 99% confidence level between species and habitats separately and species Ã habitats (p-values < 0.001, all data were given in Supporting information -Table S3).Mosses from habitat "A" in deep woodland for both species has the lowest concentrations of heavy metals.Taking the mosses deep from the forest where the area is free of pollution influences this result (S , tef anuț et al. 2021).In contrast, the collection of mosses from areas more associated with anthropogenic activities makes mosses more susceptible to pollution.For most metals for both species, this principle applies to habitat "C"wood lot.For example, only from this habitat were Cd and Pb concentrations found in both species.It should be noted that moss chemical composition is related to land use type (Di Palma et al. 2017).
The next stage of analysis was to compare the concentration of elements in mosses depending on the applied way of their preparation for further studies.Figure 1 shows the differences in element concentrations between species depending on the applied rinsing method.
Element concentrations in mosses regardless of the habitat of uptake show differences between species.The existence of a relationship, the effect of rinsing on the result of element concentration was checked.For manganese and zinc the p-value was < 0.001, for cadmium p ¼ 0.004, for lead p ¼ 0.03 and for iron p ¼ 0.02 (all data are given in Supporting information -Table S4).For copper and mercury -rinsing irrespective of moss species and habitat of its collection had no statistically significant difference.The level of significance as well as the range of elements for which the method of rinsing affected their concentrations confirms the previously obtained results ( Swisłowski, Kosior, et al. 2021).The importance of this treatment was also demonstrated by the change in CV values as a result of conditioning mosses in demineralized water compared to mosses treated only mechanically without rinsing (Table 2).The results in Table 2 indicate that in most cases the application of rinsing procedure reduced the value of CV.Only for 21.4% of the cases the application of rinsing did not reduce these values.The biggest changes decreasing the CV were observed for iron regardless of the species and sampling location.
The next step was to evaluate the relationship and correlation between the vitality of mosses (chlorophyll content and photosynthetic activity) and the concentration of heavy metals.Table 3 presents the chlorophyll a and chlorophyll b content determined in mosses collected from habitats with different levels of air pollution by heavy metals.
The results of chlorophyll content indicate its homogeneity for the species S. fallax.Lower values for D. polysetum can be observed for habitats "A" in deep woodland and "C"  the wood lot.The values for habitat "B" (forest road) are similar for the moss S. fallax.Effects of elements on photosynthetic pigments are primarily due to the degradation of damino levulinic acid dehydratase entailed in chlorophyll biosynthesis (Singh et al. 2017).The morphology and the anatomical structure of moss leaves and their chlorophyll content are related to the photosynthetic activity (Krupa 2014;2015).The results of Spearman's R non-parametric correlation statistics indicated that, depending on the particular moss species (not taking into account the moss collection habitat), there are statistically significant correlations between vitality parameters and element concentrations.For example, for the species S. fallax, a moderately strong positive correlation (0.43) with a significance level of p < 0.01 was found between the content of chlorophyll-a and the value of actual photochemical efficiency (yield).On the other hand, for D. polysetum there were many positive correlations between chlorophyll a and chlorophyll b contents in relation to concentrations of selected elements (detailed data are presented in Supporting information -Table S5).An important aspect in the context of using live mosses in active biomonitoring is measuring their vitality; this was investigated by analyzing its changes during one-month storage of mosses in the laboratory (Figure 2).
One-month storage of mosses in laboratory conditions (average temperature of 22 C and relative humidity of 40%) led to a drastic decrease in actual photochemical efficiency (yield) (Figure 2).The mean value of the decrease for S. fallax was 86.6% and for D. polysetum 79.1%.

Discussion
The use of mosses as heavy metal biomonitors has been debated for many years (Afroz 2018;Stankovi c et al. 2018;Ren et al. 2021).The concentrations of heavy metals in the mosses shown in Table 1 indicate comparably low levels of contamination as in previous studies carried out in Opolskie Voivodeship, southwestern Poland ( Swisłowski, Kosior, et al. 2021).In comparison, the results of passive biomonitoring carried out in the Polish-Czech industrial borderland differ significantly from the obtained values in this study (Motyka et al. 2020).To a large extent, element concentrations in mosses are influenced by the contribution of nearby territorial emissions and more polluted areas in southern Poland than in the north of the country (Kłos et al. 2018).Therefore, it is possible to determine local emission sources due to element concentrations deposited in mosses (Hu et al. 2020) or to determine the spatial and temporal extent of pollution (Zupan ci c and Bozau 2022).It is important to monitor elemental deposition in the environment using several moss species at once because of the possibility of comparing their accumulation capacities (Tien et al. 2020).However, it is important to consider those species with appropriate lamina cell shape and cell wall thickness -species with long and thin cells (Pseudoscleropodium purum, Hypnum cupressiforme, and Pleurozium schreberi) have been shown to be more tolerant to heavy metals than species with isodiametric cells (Plagiomnium affine and Physcomitrium patens) (Petschinger et al. 2021).Methodological aspects of ensuring quality control of individual biomonitoring steps are also important: standardization of sample collection or storage (Dołe R gowska et al. 2021).

Effects of pre-exposure preparation of mosses on active biomonitoring results
Prior to exposure, the material should be treated to increase its homogeneity (Adamo et al. 2007).Flushing, in combination with rainfall has been confirmed to influence the distribution of deposited elements in mosses (Hansson et al. 2015;Meyer et al. 2015).The influence of tree canopy and moss uptake in passive biomonitoring is usually characterized by high variability of element concentrations -CV coefficient (Parzych 2014).The washing procedure will not negatively affect the strong vitality of mosses (Tretiach et al. 2007).Due to the significant differences between element concentrations in washed and unwashed Pseudoscleropodium purum mossescombining data subjected to such treatment is not valid and impossible to compare using both methods at the same time (Fern andez et al. 2010).This was demonstrated in previous experiment ( Swisłowski, Kosior, et al. 2021), and is also confirmed by the data in this work  (Figure 1. and Table 2).However, the use of the washing procedure is dependent on the type of performed experiment, as washing samples were shown to be ineffective in determining the bioconcentration fraction (Aboal et al. 2011).Recent literature indicates that due to the very large differences in element concentration between washed and unwashed samples, this aspect should not be ignored (Fern andez et al. 2015;Aboal et al. 2017;Dołe R gowska and Migaszewski 2019;2020).The preparation of mosses before exposure is also an object of research in water quality biomonitoring (Deb en et al. 2020).The washing procedure is an essential step in the preparation of mosses before trace element analysis (Real et al. 2021).The effect of washingchloroform rinsing removes much of the copper on the plant surface (changes in % atomic weight) (Elvira et al. 2020).The washing procedure has also been used previously to remove all particles on the moss surface (Perez-Llamazares et al. 2011).Raw moss can be soaked for several hours and can be implemented to remove additional sand and dust (Hossain 2020).As mentioned earlier, the washing procedure is essential prior to moss exposure under the moss-bag technique ( Swisłowski, Kosior, et al. 2021).Washing mosses may not always be effective in getting rid of all particulate matter on their surface, but the variability in element concentrations in mosses prior to exposure is reduced (Figure 1. and Table 2).A washing of metals from the moss tissue was not detectable, suggesting active binding of metals from the atmosphere to moss tissue (Sabovljevi c et al. 2020).The higher zinc and manganese concentrations after washing observed in samples have been reported previously (Dołe R gowska and Migaszewski 2020).According to the authors, this is related to the fact that the affinity of these elements for extracellular binding sites is lower than that of other studied elements, thus they can be absorbed into moss tissues from particles deposited on their surface.
The homogeneity of the material provides more reliable and accurate information about the contaminants that mosses accumulate during exposure.In turn, the storage process of mosses itself affects the element concentrations (P.schreberi mosses, 7 days of storage) (Dołe R gowska and Migaszewski 2020).A recent report contradicts this, and for Fontinalis antipyretica moss, no changes in trace element concentration were found for the three storage procedures tested (14 days) (Villares et al. 2023).Along with other procedures, such as moss habitat conditions and exposure time, washing is an important part of moss preparation (Rogova et al. 2018;2021).These examples demonstrate the validity of adequate, consistent, and uniform preparation of mosses for display within the moss-bag technique after they have been collected in the forest.

Effects of environmental factors on moss vitality
Selected environmental factors including species, season, time of day and water availability have a significant effect on photosynthetic activity (We Rgrzyn et al. 2021).Literature examples indicate the importance of including moss vitality measurements in biomonitoring studies (Capozzi et al. 2020;Boquete et al. 2021;Sossey Alaoui et al. 2021;Swisłowski et al. 2021a;et al. 2021b).Actual photochemical efficiency (yield) below 0.1 is the critical value below which the moss is only a natural sorbent and not a living biomonitor because it loses its viability (Lichtenthaler et al. 2005;Laisk et al. 2014).For the moss Leptodictyum riparium there was a steady decrease of photosynthetic efficiency (Fv/Fm) correlated with the heavy metal concentrations (Maresca et al. 2022).For the same species, its exposure under the moss bag technique resulted in toxic metal pollution caused severe ultrastructural damage in moss (Esposito et al. 2018).Cadmium negatively affects by decreasing total chlorophyll content in bryophytes (Bellini et al. 2021).Chlorophyll content was found to decrease during exposure of mosses in urban areas without a close relationship with heavy metal concentrations ( Swisłowski et al. 2020).Exposure to the pollutants has a dose-dependent inhibitory effect on photosynthetic attributes or pigments (Gupta and Seth 2021;Kumar et al. 2023).Relationship between elemental composition of mosses and micro-and macroscopic morphological traits was proven -Pb, Cd, and Cu showed a negative correlation with leaf form and density and Cu, Fe or Mn were clearly negatively related to leaf length and surface area (Fern andez-Mart ınez et al. 2021).Cd seems to be the most toxic in terms of chlorophyll-a content reduction for Barbula consanguinea, Hyophila apiculata, and H. involute (Phaenark et al. 2022).However foliar supplementation with a-lipoic acid mitigates the toxicity of Cd on photosynthetic pigments and can represent a sustainable means of improving the quality of plants grown on metal affected soils (Yadav et al. 2022).
Low water availability [or lack thereof as in the case of desiccation under laboratory temperature conditionsdistribution measured as mass loss in the laboratory to about 60% mass loss in S. fallax (Bengtsson et al. 2016)] results in reductions in photosynthesis and chlorophyll content for species of all Sphagnum species in both in situ and laboratory experiments.These trends are explained by the inhibition of cellular metabolism, the inhibition of biochemical processes or the decrease in the internal N content (Chiapusio et al. 2022).For the moss Pseudocrossidium replicatum, already after seven days of dehydration under laboratory conditions, Fv/Fm was undetectable (R ıos-Mel endez et al. 2021).For results shown in Figure 2, we observed a negative effect on the changes in actual photochemical efficiency (yield) due to the monthly storage of mosses in the laboratory.The use of such material disqualifies it for use in active biomonitoring of urbanized areas.

Conclusions
The variability in element concentrations deposited in mosses is the lowest for the material sampled in deep forest, unpolluted areas.Thus these specimens are best selection for further processing in the moss-bag approach.
The rinsing procedure for moss samples with homogenization uniform the material prior to exposure (CV for majority metals <10%) which was confirmed by statistical analysis and indicates its versatility regardless of the species and its collection habitat.
One-month storage of mosses in laboratory conditions led to a decrease in actual photochemical efficiency over 80%.This eliminates such material as a living bioindicator of air pollution in the moss-bag technique application.
In the future, more attention during biomonitoring experiments should be paid to optimizing and standardizing the prepared material related to the measurement of the viability of bioindicators.

Figure 1 .
Figure 1.Concentrations of elements in S. fallax (Sp) and D. polysetum (Dp) depending on the way of preparation: white box (unrised) and grey box (rinsed mosses).

Figure 2 .
Figure 2. Changes in actual photochemical efficiency (yield) during moss storage.Bars (n ¼ 12) indicate mean values with standard deviation (whiskers).White bars: actual photochemical efficiency (yield) in the forest before collecting, and black bars: mosses after one month stored under laboratory conditions.Different lowercase letter(s) represent significant differences (Duncan's multiple range test, p < 0.05).
). Mosses were collected in the Swietokrzyskie Voivodeship in southeastern Poland -Sta ˛pork ow Forest District in July 2021.Examples of biomonitoring research carried out in the Swietokrzyskie Voivodship used different biomonitors (e.g., mosses, lichens, needles, or algae)
Note: A habitatin deep woodland; B habitaton a forest road; C habitatthe wood lot.n.d.: no dataconcentrations below the limit of quantification of the AAS analytical method used

Table 2 .
Comparison of CV values for mosses prepared for analysis in accordance with unrinsed (left site of column) and rinsed method (right site of column) [%].concentrations below the limit of quantification of the AAS analytical method used or the number of samples did not allow the determination of CV; bold values mean the lowest value for rinsed way in relation to unrinsed samples of mosses.
Note: A habitatin deep woodland; B habitaton a forest road; C habitatthe wood lot.n.d.: no data -

Table 3 .
Mean chlorophyll concentrations (± standard deviation) in mosses collected from analyzed habitats (mg/L).Note: A habitatin deep woodland; B habitaton a forest road; C habitatthe wood lot.