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Mercury contamination is a global environmental problem. This pollutant is highly toxic and persistent which makes it extremely susceptible to biomagnify, i.e. increase its concentrations as it moves up the food chain, reaching levels that threaten wildlife and, ultimately, ecosystems’ function and structure. Mercury monitoring is thus crucial to determine its potential to damage the environment. In this study, we assessed the temporal trends of the concentrations of Hg in two coastal animal species closely connected by a predator-prey interaction, and evaluated its potential transfer between trophic levels using the δ15N signatures of the two species. For this, we performed a multi-year survey of the concentrations of total Hg and the values of δ15N in the mussel Mytilus galloprovincialis (prey) and the dogwhelk Nucella lapillus (predator) sampled along ∼1500 km of the North Atlantic coast of Spain over a 30-year period (five surveys between 1990 and 2021). Concentrations of Hg decreased significantly between the first and the last survey in the two species studied. Except for the 1990 survey, the concentrations of Hg in mussels were amongst the lowest registered in the literature for the North East Atlantic Ocean (NEAO) and the Mediterranean Sea (MS) between 1985 and 2020. Nonetheless, we detected Hg biomagnification in almost all surveys. Worryingly, trophic magnification factors obtained here for total Hg were high and comparable to the found in the literature for methylmercury, the most toxic and readily biomagnified form of this element. The δ15N values were useful to detect Hg biomagnification under normal circumstances. However, we found that nitrogen pollution of coastal waters differentially affected the δ15N signatures of mussels and dogwhelks limiting the use of this parameter for this purpose. We conclude that Hg biomagnification could constitute an important environmental hazard even when found at very low concentrations in the lower trophic levels. Also, we warn that use of δ15N in biomagnification studies when there is some underlying nitrogen pollution problem might lead to misleading conclusions Authors belong to the Grupo de Referencia Competitiva GRC GI-1252/GPC2020–23 (ED431C 2020/19) which is co-funded by Xunta de Galicia and ERDF (EU) SI

Polycyclic Aromatic Hydrocarbons (PAHs) are an important class of pollutants which mostly come out from incomplete combustion of organic materials including fossil fuels. For this reason, they are often found at high concentrations in cities, contaminating air with their gas and particle phase. While European Union policies try to reduce their concentrations, huge efforts are still devoted to mitigate the pollution by PAHs. One such measure of mitigation is the use of plants in capturing PAHs and other chemicals due to the ability of leaves in filtering both gas and particle fractions. In this work thirteen conifer species among those frequently living in temperate and Mediterranean cities were selected and concentrations of 16 PAHs were measured in needles. The data for spruce were used to back-calculate air concentrations of PAHs which were later used to obtain specific equations of plant-air partition coefficient (K

Seaweeds are dominant organisms in coastal environments. However, in the context of global change, the integrity of these organisms is threatened by metal pollution. It is therefore important to understand how seaweeds are affected by metal concentrations in the water. Measuring the concentrations of metals in seaweed provides information about the effects of metal pollution on the seaweeds themselves and their ecosystems. Nonetheless, correct interpretation of this type of analysis requires knowledge of the cellular location of the pollutants, as the effects will differ depending on whether the metals are present in particles adhered to the surface, attached to external polysaccharides or dissolved in the cytoplasm. Thus, the objectives of this study were to compile the available information on the subcellular distribution of metals in seaweeds and to conduct a critical review of the information. We found that the existing studies provide contrasting, sometimes contradictory, results. Thus, metals have been detected entirely intracellularly and also mainly outside of the cells. In all of the studies reviewed, which used different techniques (mainly extracellular elution, X-ray microanalysis and centrifugation), methodological and/or conceptual problems were identified that raise questions about the effectiveness of each approach. To obtain reliable information about the distribution of metals in algal cells, further studies must be conducted that take into consideration the differences between elements and algal species and the limits of the methods used to measure the elements.

Although soil is known to contribute to the concentrations of elements in moss, protocols for atmospheric biomonitoring with terrestrial moss do not include recommendations on how to address this factor. As a result, researchers indiscriminately use a wide range of detection/correction methods without considering whether the results are equivalent. In this study, three of these methods were compared: i) use of the enrichment factor (EF) index; ii) calculation of the ratios of different elements in soil and moss, and subtraction of the contribution of soil concentrations from the raw concentrations of elements in mosses (SCS); and iii) positive matrix factorization (PMF), a receptor modelling method for source apportioning based on multivariate analysis techniques. The aim of the comparison was to determine whether the methods produce equivalent results and, if not, which method is the most appropriate for use in moss biomonitoring surveys. The data used corresponded to 146 samples of Pseudoscleropodium purum collected from a regular sampling grid of 15 × 15 km in Galicia (NW Spain). Comparison of the methods revealed that, although they yield relatively similar results, the corresponding interpretations are not equivalent and none of the methods provides a reliable estimate of the soil contribution to the concentrations of elements in moss samples. Independently of the technique applied, use of Ti as a reference element is not recommended, because, at least in this study, it was present at unusually high levels in moss. Given the absence of a reliable correction method and the fact that most elements are present in fairly high amounts in the soil, we recommend using atmospheric biomonitoring with moss only for Cu, Zn and Cd, i.e. for those elements in moss for which the soil contributes very low amounts and corrections are not therefore necessary The authors belong to the Galician Competitive Research Group GRC/GPC2016-002 and to the CRETUS Strategic Partnership (AGRUP2015/02) which are co-funded by FEDER (EU). Authors would like to thank RIAIDT-USC for the use of analytical facilities. P. Giráldez is grateful to the Spanish Ministerio de Ciencia, Innovación y Universidades for a grant awarded within the Programa de Formacion de Profesorado Universitario (FPU 2018 [grant number FPU18/04134]). Z. Varela was supported by a postdoctoral research grant awarded by the Autonomous Government of Galicia (Spain) SI

Tree canopies play a key role in the cycling of polycyclic aromatic hydrocarbons (PAHs) in terrestrial ecosystems, as leaves can capture PAHs from the air. In this study, accumulation of PAHs was compared in an evergreen species, P. pinaster , and in a deciduous species, Q. robur , in relation to some physio-morphological characteristics. For this purpose, pine needles and oak leaves collected from different sites across Galicia (NW Spain) were analysed to determine PAH contents, specific leaf area, stomatal density and conductance. Leaves and needles contained similar total amounts of PAHs. The major contribution of particle-bound PAHs in oak (the concentrations of 4- and 5-ring PAHs were two times higher, and those of 6-ring PAHs five times higher in oak than in pine) may be related to the higher specific leaf area (13 and 4 cm 2 g −1 dry mass in respectively oak and pine). However, the major contribution of vapor-phase PAHs in pines may be affected by the stomatal conductance (two times higher in pine than in oak). Moreover, an increase in the diameter at breast height of trees led to an increase in accumulation of PAHs, with pine capturing higher amounts of low and medium molecular weight PAHs. The study findings underline the potential role of trees in improving air quality, taking into account the canopy biomass and life cycle.

In this study, we used amplified fragment length polymorphism analysis to investigate the genetic structure of the terrestrial moss Pseudoscleropodium purum (Hedw.) M. Fleish. naturally exposed to different levels of atmospheric deposition of heavy metals. We also determined the heavy metal concentrations in samples of this moss to evaluate whether there was a relationship between atmospheric pollution and population genetic diversity. A low level of genetic diversity and a limited gene flow among populations were observed which is in accordance to the prevalence of asexual reproduction in this species. In addition, no significant correlation was found between metal content and gene diversity in P. purum, probably because of the common history of the sampled populations and/or to the lack of a drastic reduction of the size of the population; nonetheless, a clear genetic structure was evident in relation to the existing pollution gradient. Thus, based on the results of the principal coordinate analysis and Bayesian analysis of the genotypes, the mixed structure of the second most polluted population would suggest an ongoing differentiation of metal-tolerant genotypes in the most polluted sites of the sampling area.

Protein expression was assessed in samples of Pseudoscleropodium purum cross-transplanted between one unpolluted (UNP) and two polluted (POLL) sites. Firstly, the level of expression (LE) of 17 proteins differed between native mosses from both types of sites, but differences were only maintained throughout the experiment for 5 of them. The LE of these five proteins changed over time in mosses transplanted from UNP to POLL and vice versa, becoming similar to that in autotransplants. However, these changes occurred slower than changes in the heavy metal concentrations measured in the same samples, and therefore they were not related to atmospheric pollution. Although the proteins identified were associated with moss metabolism, the expected growth reduction in samples autotransplanted within POLL (as a result of the down-regulation of photosynthesis-related proteins), did not occur. This supports the hypothesis that mosses growing in polluted areas adapt to heavy metal pollution and are able to reduce/overcome their toxic effects (i.e., reduced growth). Nevertheless, further specific research must be carried out to identify the proteins involved in this type of response, as lack of information on the bryophyte genome precludes us from reaching further conclusions.

We measured the concentrations of Cd, Cu, Hg, Pb and Zn in samples of the terrestrial moss Pseudoscleropodium purum reciprocally transplanted between an unpolluted and two polluted sampling sites. At the beginning of the experiment, the concentrations of all these elements differed significantly between mosses from the unpolluted site and mosses from the polluted sites. In general, the concentrations of the heavy metals in mosses from both polluted sites transplanted to the unpolluted site decreased until they reached the same levels as in autotransplants at this site (after 480-840 days). However, the concentrations of all heavy metals in mosses transplanted from the unpolluted site to both polluted sites increased to higher levels than in the autotransplants (except for Cu, Hg and Pb at one of the sampling sites). These results led us to conclude that mosses that have been continuously exposed to high atmospheric deposition of heavy metals undergo an adaptive response (probably genotypic) to such conditions. We therefore recommend active rather than passive biomonitoring of air quality in industrial environments because atmospheric deposition could be underestimated, and also recommend further investigation into the mechanisms involved in this response.

The concentration of Pb was determined in feathers (contour feathers: mantle, pectoral, ventral, and primary- and secondary-coverts) of two sedentary species of raptors in Galicia (NW Spain): the tawny owl (Strix aluco) and the Northern goshawk (Accipiter gentilis). A high degree of intraindividual variability was observed in all types of feathers, with coefficients of variation exceeding 100 percent. The correlations between feather types were too low to enable use of a single type of body feather to predict the concentration of Pb in the other feathers. The number of body feathers required to differentiate individuals on the basis of the concentration of Pb was extremely high, in some cases higher than the number of the particular type of feather in the bird. All of this provides clear evidence that the contour and covert feathers of the raptor species considered cannot be used to biomonitor contamination by Pb, at least in this sample where the overall feather concentration were fairly uniform.

The concentrations of Hg were determined in all primary flight feathers from 20 specimens of Buteo buteo and 49 specimens of Accipiter gentilis, which had died in various Wildlife Recovery Centres in Galicia (NW Spain) between 2001 and 2009. The aim of the study was to standardize the feather or feathers that should be analyzed in each species to enable inter-individual comparison of the levels of contamination, taking into consideration any variability in the concentrations of Hg caused by the birds' molting patterns. For both species the results show a high degree of both inter (e.g. an individual of A. gentilis showed 426 ng g(-1) of Hg as median and 568 ng g(-1) as median absolute deviation, MAD) and intra-individual variability (e.g. primary feather number seven - P7 - of all the A. gentilis individuals showed 537 ng g(-1) of Hg as median and 1129 ng g(-1) as MAD). Although molting did not have a clear effect on the concentrations of Hg in the flight feathers, it was possible to identify the most representative feather in each individual, taking into account the amount of metal excreted in each feather and the intra-individual variability. For B. buteo use of flight feather P6 is recommended for Hg biomonitoring studies; the amount of Hg excreted to this feather was 509 ng as median, which represents the 14% (from 10 to 20%) of the total Hg present in all the primary feathers. For A. gentilis the recommended feather is P7, which has a median of 86 ng of Hg (corresponding to 12%, and varying between 5 and 22%).

The use of bryophytes and lichens as biomonitors of atmospheric contamination, particularly contamination by heavy metals, is a well-known and widely applied technique. Determination of the total concentrations in organisms is often used in such studies, but has some disadvantages that may be resolved by using the sequential elution technique (SET). This technique involves successive washing steps that enable quantification of the elements in the different cell compartments (intercellular, extracellular, and intracellular), and finally determination of the remaining fraction, i.e. particulate material. The key step in the SET is the correct extraction of the extracellular fraction, for which a suitable extractant must be used for each different element considered. We have found only seven studies that have focused on selecting suitable extractants, which may be metal cations or chelating agents. Ethylenediaminetetraacetic acid is presented as the most appropriate extracellular extractant for Al, C...

The sequential elution technique (SET) is used to determine the distribution of elements in the different cell fractions of mosses. The extracellular extractants most commonly used in this technique are NiCl2 and EDTA, although there are certain disadvantages associated with their use. In order to avoid such problems, we searched for new extractants that displace extracellularly bound metals, either because they are present at high concentrations (Ca) or because they have a high affinity for cation exchange sites (Hg and Au). The compounds HCl, NaCl, and CaCl2 were tested as extractants of the former type, as possible alternatives for the determination of extracellular metals in the moss Pseudoscleropodium purum. Calcium chloride was finally chosen as is it potentially the most successful in terms of binding to the cation exchange sites without altering the membrane permeability. The concentration chosen was 160 mM, as this yielded maximum displacement of Zn without membrane alterations. An experiment was then carried out to test the efficiency of Ca in extracting extracellular Zn, under laboratory and field conditions. In addition, Hg and Au were tested, at different concentrations, as extractants with high affinity for cation exchange sites, as neither of these elements has previously been used in the SET, and both display electronic characteristics that suggest their potential usefulness in displacing other cations from cation exchange sites. The results obtained show that extraction of extracellularly bound metal by high concentrations of Ca should be ruled out, as total extraction of Zn was not achieved. Both Hg and Au produced membrane alterations at low concentrations and moreover, neither was more efficient at extracting Zn than the reference extractant (20 mM NiCl2).

Three experiments were carried out to select the best extractant for use in the sequential elution technique, to enable extraction of Cu, Pb, V and Zn from the extracellular fraction of the terrestrial moss Pseudoscleropodium purum. The optimal concentrations of the extractants tested (CoCl2, NiCl2, Pb(NO3)2, SrCl2, dimercaprol, EDTA, penicillamine) were determined on the basis of the maximum extraction of Zn achieved without any alteration of the plasma membrane. The capacity of these agents (at the optimal concentrations established) to extract the extracellular fractions of Cu, Pb, V and Zn was then evaluated. Extraction with 10 mM EDTA is recommended for all 4 elements considered. As a second option, the use of 50 mM penicillamine is recommended to extract Cu, 30 mM dimercaprol to extract Pb and V and 20 mM NiCl2 to extract Zn. It was also concluded that these results cannot be extrapolated to other cryptogams, and that separate assays are required.

The effects of growth of autoirrigated, shaded transplants of Pseudoscleropodium purum on the quantification of tissue concentrations of Cd, Cu, Hg, V and Zn, were investigated in 4 exposure periods, each of 56 days, at 7 sampling sites (contaminated and uncontaminated). Concentrations of the elements in the basal portions of the moss shoots were compared with the concentrations in the portions of the shoots that grew during the exposure period. Mercury and V were present at lower concentrations in the new portions of the shoots than in the basal portions, whereas the opposite was true for Cd, Cu and Zn. The magnitude of error introduced by growth was not negligible, and in some cases was higher than 40%, relative to the results obtained by analysis of the whole shoot. Devitalization of moss prior to its use as transplant material is recommended to avoid growth of the plant during the exposure period.

A new simple method is proposed for identifying sources of small scale pollution, by determining the tissue concentrations of pollutants in terrestrial mosses. The method is based on spatial characterization of the processes that generate accumulation of pollutants in these organisms. The steps involved in implementing and applying the method are: (i) obtaining data on pollutants in moss samples collected at least at 35 pairs of sampling sites (SS) separated by 1 km distance; (ii) study of the distribution of the differences in concentration between the pairs of SS, eliminating pairs affected by foci that generate small scale processes; (iii) characterization of the resulting normal distributions and calculation, for different levels of significance, of the probability of a value lying within these distributions, and (iv) application of the calculated quantiles to data corresponding to the differences in concentrations between pairs of SS in the vicinities of foci of small scale pollution, to determine which elements can be considered as pollutants. Given the small number of data required once the distribution of the differences has been established, the method is an inexpensive, efficient way of establishing the probability that a pollutant is being emitted from a particular source. The method is very useful for verifying the results of pollution inventories, evaluating new technologies and improving the design of regional networks for biomonitoring of air quality with terrestrial mosses.

Plants are used as target organisms in most biomonitoring studies of atmospheric deposition of dioxins and furans (PCDD/F) because the most common route of entry of these pollutants into ecosystems is through soils and plants. However, comparison of the results of such studies is difficult because different plant species are used. In the present study we propose the use of mosses as biomonitors of PCDD/F deposition because of their suitable characteristics (absorption of substances from the atmosphere only, large absorbing surface, wide distribution of some species, etc.). We measured the PCDD/F concentrations in tissues of Pseudoscleropodium purum collected in areas far from known sources of pollution and in the surroundings of potential sources of these pollutants (several types of industries, incinerators and burning rubbish dumps). Part of the samples were collected with the aim of monitoring changes in the pollution intensity around some selected sources over time, others were collected to study the spatial gradient of pollution around other sources. Our results indicate that the measurements of the concentration of PCDD/F in the moss tissue allow the detection of strong and weak pollution sources and that these measurements are sensible enough to monitorize changes in the pollution intensity along time, as well as to describe the spatial gradients of pollution created around point sources, and the differences in the relative abundance of isomers (homologue profiles) among different sources. We concluded that mosses are as good biomonitors for PCDD/F as they are for other types of pollutants.

As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world’s climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture (Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S.palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research. Electronic supplementary material The online version of this article (doi:10.1007/s11240-014-0658-2) contains supplementary material, which is available to authorized users.

The total concentrations and the extra- and intracellular concentrations of Ca, Cd, K and Zn were determined in samples of the moss Pseudoscleropodium purum, collected every 3–4 days at two sampling sites (one located in a contaminated zone, n = 101, and another in an uncontaminated zone, n = 62). The medians for the total concentrations and the concentrations in the different cell compartments varied widely over time in for the elements studied. In addition, the variability was always lower for the total concentrations, being usually the highest for the extracellular concentrations. For metabolically important elements (i.e. Ca and K), the temporal variability was due to variation in the concentrations of these elements in the extracellular fraction caused by environmental factors. For Zn, the variation was caused by the presence of metal bound to particles on the moss surface and its effect on the extracellular fraction, whereas for Cd, the intracellular concentration was independent of the total concentration. The results enabled us to conclude that neither of the cell compartments considered integrates the studied contaminants, showing a high temporal variation along time, especially for the extracellular location.

Multivariate analysis of environmental data sets requires the absence of missing values or their substitution by small values. However, if the data is transformed logarithmically prior to the analysis, this solution cannot be applied because the logarithm of a small value might become an outlier. Several methods for substituting the missing values can be found in the literature although none of them guarantees that no distortion of the structure of the data set is produced. We propose a method for the assessment of these distortions which can be used for deciding whether to retain or not the samples or variables containing missing values and for the investigation of the performance of different substitution techniques. The method analyzes the structure of the distances among samples using Mantel tests. We present an application of the method to PCDD/F data measured in samples of terrestrial moss as part of a biomonitoring study.

Compositional analysis consists of a group of techniques used to manipulate closed (=compositional) data, i.e. multivariate data summing to a fixed quantity (proportions, percentages). It is based on the analysis of the relations among variables and the use of logarithmic transformations. It has been claimed that this group of techniques should be used to analyse profiles of pollutant sources because profiles themselves are proportions. We show in this paper that, for the exploratory analysis of these data, a good strategy is to combine the analysis done with and without transformation because they give different and complementary insights on the structure of the data. We discuss in particular the study of processes such as the mixing of pollutants produced by different sources and the exponential decay of concentrations with distance to the source found in many studies. The clr transformation is also appropriate for the study of the variables having small proportions, which remain concealed by the abundant variables when analysed without transformation. We present simulations to illustrate these ideas and we also apply these techniques to two data sets of PCDD/F content in moss tissues.