J. Ángel Fernández

Assessing the tolerance of the terrestrial moss Pseudoscleropodium purum to high levels of atmospheric heavy metals: a reciprocal transplant study.

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 sequential elution technique applied to cryptogams: a literature review

Abstract 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, Co, Cu, K, Mg, Pb, V, and Zn, for which it has been tested, and for Cd and Fe, for which it has not yet been tested (although it is known to be capable of extracting these elements). The only extractant that is capable of extracting extracellular Hg is dimercaprol. The technique has been used in laboratory studies and less often in field studies carried out in the surroundings of focal points of contamination. The elements analysed include nutrients (e.g. Ca, K, and Mg) and heavy metals (e.g. Cd, Zn, and Cu), usually associated with sources of emission of contaminants. Several problems have arisen in the application of the SET, some of which must be resolved. These include methodological problems (e.g. variability in the extraction process) and problems inherent in the SET itself, which are more difficult to resolve. Other techniques, such as histochemical techniques and electron microscopy with microanalysis, must therefore be used simultaneously with the SET to determine if the uptake of metal is only extracellular. At present, in light of the disadvantages of the quantification of the extracellular fraction, measurement of the intracellular fraction is presented as the best option because it is not affected by the metal burden in the particles, enables evaluation of environmental risks, better represents the average conditions of contamination, and enables better evaluation of phytotoxicity.

Evaluation of cations and chelating agents as extracellular extractants for Cu, Pb, V and Zn in the sequential elution technique applied to the terrestrial moss Pseudoscleropodium purum

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 (CoCl(2), NiCl(2), Pb(NO(3))(2), SrCl(2), 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 10mM EDTA is recommended for all 4 elements considered. As a second option, the use of 50mM penicillamine is recommended to extract Cu, 30 mM dimercaprol to extract Pb and V and 20 mM NiCl(2) to extract Zn. It was also concluded that these results cannot be extrapolated to other cryptogams, and that separate assays are required.

Metal and proton adsorption capacities of natural and cloned Sphagnum mosses.

Terrestrial mosses are commonly used as bioindicators of atmospheric pollution. However, there is a lack of standardization of the biomonitoring preparation technique and the efficiency of metal adsorption by various moss species is poorly known. This is especially true for in vitro-cultivated moss clones, which are promising candidates for a standardized moss-bag technique. We studied the adsorption of copper and zinc on naturally grown Sphagnum peat moss in comparison with in vitro-cultivated Sphagnum palustre samples in order to provide their physico-chemical characterization and to test the possibility of using cloned peat mosses as bioindicators within the protocol of moss-bag technique. We demonstrate that in vitro-grown clones of S. palustre exhibit acid-base properties similar to those of naturally grown Sphagnum samples, whereas the zinc adsorption capacity of the clones is approx. twice higher than that of the samples from the field. At the same time, the field samples adsorbed 30-50% higher amount of Cu(2+) compared to that of the clones. This contrast may be related to fine differences in the bulk chemical composition, specific surface area, morphological features, type and abundance of binding sites at the cell surfaces and in the aqueous solution of natural and cloned Sphagnum. The clones exhibited much lower concentration of most metal pollutants in their tissues relative to the natural samples thus making the former better indicators of low metal loading. Overall, in vitro-produced clones of S. palustre can be considered as an adequate, environmentally benign substitution for protected natural Sphagnum sp. samples to be used in moss-bags for atmospheric monitoring.

Evaluation of different contour feather types for biomonitoring lead exposure in Northern goshawk (Accipiter gentilis) and tawny owl (Strix aluco)

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.

Assessing the effects of heavy metal contamination on the proteome of the moss Pseudoscleropodium purum cross-transplanted between different areas.

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.

Substituting missing data in compositional analysis

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.

The analysis of pollutant profiles by means of compositional analysis. An application to PCDD/F data

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.

Temporal trends in mercury concentrations in raptor flight feathers stored in an environmental specimen bank in Galicia (NW Spain) between 2000 and 2013

Temporal trends in Hg concentrations were investigated in primary flight feathers from 319 specimens of three birds of prey: P7 in the northern goshawk (Accipiter gentilis), P6 in the common buzzard (Buteo buteo) and P5 in the tawny owl (Strix aluco). The samples were stored in a regional environmental specimen bank and belonged to specimens which died between 2000 and 2013 in Galicia (NW Spain). We would expect to see a decline in Hg concentrations across the study period, as data of atmospheric emissions show a gradual reduction of this pollutant in Europe in the last two decades. The study did not reveal any temporal pattern in Hg concentrations of feathers in any of the three species for the study period, may be due to the persistence of Hg in the environment, but showed a low level of contamination by this metal in the study area. In addition, the results show high intra-specific, as well as, inter-annual and inter-specific variability in data, mainly attributed to the level of exposure of the raptors to this pollutant and to the biomagnification process of Hg through food chains. These findings indicate that the high variability can be a limiting factor in the use of raptors for biomonitoring temporal patterns of Hg, but nevertheless, the technique provides qualitative information about the amount of Hg that reach the top of the terrestrial food chains.

Genetic structuring of the moss Pseudoscleropodium purum sampled at different distances from a pollution source

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.