Analysis of intra-thallus and temporal variability of trace elements and nitrogen in Fucus vesiculosus: Sampling protocol optimization for biomonitoring.

To advance the methodological standardization of the biomonitoring technique using macroalgae, we comprehensively characterized the intra-thallus and temporal patterns of variation in concentrations of a wide set of elements (Al, As, Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb, Zn, N) and δ15N signal in 6 consecutive Fucus vesiculosus apical dichotomous sections collected monthly over a four-year period (2015-2019) at 3 sites on the NW coast of Spain. The concentrations of Al, Co, Fe, Ni, Pb and Zn increased significantly from the youngest to the oldest dichotomies regardless of the sampling time and collection site; As, Cd, N and δ15N showed the opposite trend. Time series analysis revealed a significant and consistent seasonal variation of As, Cd, Co, Cu, Fe, Hg, Ni, Zn, N and δ15N concentrations, with maximum values in winter and minimum values in summer. We discussed the possible mechanisms driving these two sources of variation, and proposed an efficient and effective sampling strategy to minimize their impact in the results of biomonitoring studies, in which the part of the algal thallus selected for chemical analysis and the sampling frequency were carefully considered. This protocol will improve the conclusions and comparability of biomonitoring data from coastal environments.

Matching times: Trying to improve the correlation between heavy metal levels in mosses and bulk deposition.

The relationship between the concentrations of metals in moss tissues and atmospheric deposition is highly complex, resulting in a general lack of correlations between these two matrices. Here, we tried to improve the significance of the moss-bulk deposition (BD) relationship by eliminating the mismatch between the time that the moss tissue selected for analysis is exposed to atmospheric deposition, and the time during which BD is collected. For this, we analysed the concentrations of Cd, Hg and Pb in new grown tissue of Pseudoscleropodium purum and BD collected monthly, for one year, in 21 sampling sites (SS) under different degrees of pollution. Additionally, we assessed how different moss tissues, including native moss (green parts and new grown tissues of P. purum) and moss transplants of Sphagnum denticulatum, reflect BD to find out which moss tissues provide a better estimate of the atmospheric deposition of heavy metals. First of all, our results showed that eliminating the mismatch between native moss exposure time and BD collection period is not enough to improve their correlation. Environmental variation emerged as the main driver of tissue content variation altering the moss-BD relationship unpredictably. Secondly, native P. purum represents BD values better than devitalized transplants by displaying a greater number of significant correlations with BD. Specifically, green parts of P. purum generally represent better BD than new grown tissues. Overall, we conclude that neither native mosses nor transplants are good estimators of atmospheric heavy metal deposition rates. However, they are good qualitative indicators of the atmospheric deposition, by allowing us to differentiate SS subject to a wide range of pollution levels. Additionally, green parts of P. purum, and likely of other mosses with similar growth forms, should be used in passive biomonitoring studies to make results from different studies comparable.

Sampling optimization for biomonitoring metal contamination with marine macroalgae.

The aim of the present study was to optimize the protocol for sampling marine macroalgae to be used to biomonitor heavy metal contamination in marine ecosystems. For this purpose, we collected 50 subsamples of the brown seaweed Fucus vesiculosus at random in each of three sampling sites (SS) and determined the concentrations of Al, As, Cd, Co, Cr, Cu, Fe, Hg, N, Ni, Pb, Zn and δ15N. We used semivariograms to explore the possible existence of spatial structure in the concentrations of the elements. Spatial structure was observed in 88% of the semivariograms studied, with element concentrations varying longitudinally and transversally along the SS. Using randomization techniques, we estimated that in each SS, a minimum of 30 evenly distributed subsamples should be collected within three bands parallel to the coastline (and also at different heights on the rocks if necessary), and analyzed in a single composite sample representative of the intra-SS variability.

Application of macroalgae analysis to assess the natural variability in selected pollution concentrations (N and Hg), and to detect sources of it in coastal environments.

Here we present a new method in which algae are used to detect sources of heavy metal and organic pollution in coastal areas. The procedure involves characterization of the natural range of concentrations of the elements in areas not affected by local sources of pollution and subsequent comparison of concentrations in the study site to these natural range levels. To develop the method, the concentrations of various elements were determined in specimens of the macroalgae Fucus vesiculosus collected at >150 sampling sites along the shoreline. The natural variability in the element concentrations in these zones was established by determining the differences in the tissue concentrations of the elements between pairs of samples separated by different distances. The method was then tested in the surroundings of possible sources of nitrogen and was found to be a tool for detecting sources of small scale nitrogen contamination and for monitoring and evaluating water quality.

Biomonitoring coastal environments with transplanted macroalgae: A methodological review.

The use of macroalgae transplants is a recent technique used in pollution biomonitoring studies in marine ecosystems. Only 60 articles published between 1978 and 2017 reported the use of this environmental tool for the active biomonitoring of inorganic pollutants and nutrients worldwide. In this review paper, we evaluated studies on this topic in relation to the development of methodological aspects of the technique and the degree of standardization of the protocols used. On the basis of findings of this review, we conclude that the technique is not yet standardized and that uniformisation of protocols is required to enable comparison of the results of different studies. We propose a new protocol for applying the technique, in which each suggestion has been carefully and rigorously compared with the relevant findings reported in the available literature.

Do mosses exist outside of Europe? A biomonitoring reflection.

The passive moss biomonitoring technique has been proved a useful environmental tool for the study of the air quality. However, after more than 40years of its discovery, it has not been used yet in decision making when dealing with atmospheric pollution. Scientific efforts and funding are wasted when these sort of findings do not have a meaningful impact on society. Thus, the aim of this review is to showcase the reasons preventing the worldwide application of the moss technique. The results showed that the possible reasons underlying this problem are the lack of standardization of the technique, transmission of a false idea of robustness, and the lack of a theoretical background. Knowing and accepting these problems is the first step to encourage scientists and funding bodies to invest their efforts in really improving the technique for its application in environmental policies and not only in scientific circles.

Quantification of the overall measurement uncertainty associated with the passive moss biomonitoring technique: Sample collection and processing.

In this study we examined 6080 data gathered by our research group during more than 20 years of research on the moss biomonitoring technique, in order to quantify the variability generated by different aspects of the protocol and to calculate the overall measurement uncertainty associated with the technique. The median variance of the concentrations of different pollutants measured in moss tissues attributed to the different methodological aspects was high, reaching values of 2851 (ng·g-1)2 for Cd (sample treatment), 35.1 (µg·g-1)2 for Cu (sample treatment), 861.7 (ng·g-1)2 and for Hg (material selection). These variances correspond to standard deviations that constitute 67, 126 and 59% the regional background levels of these elements in the study region. The overall measurement uncertainty associated with the worst experimental protocol (5 subsamples, refrigerated, washed, 5 × 5 m size of the sampling area and once a year sampling) was between 2 and 6 times higher than that associated with the optimal protocol (30 subsamples, dried, unwashed, 20 × 20 m size of the sampling area and once a week sampling), and between 1.5 and 7 times higher than that associated with the standardized protocol (30 subsamples and once a year sampling). The overall measurement uncertainty associated with the standardized protocol could generate variations of between 14 and 47% in the regional background levels of Cd, Cu, Hg, Pb and Zn in the study area and much higher levels of variation in polluted sampling sites. We demonstrated that although the overall measurement uncertainty of the technique is still high, it can be reduced by using already well defined aspects of the protocol. Further standardization of the protocol together with application of the information on the overall measurement uncertainty would improve the reliability and comparability of the results of different biomonitoring studies, thus extending use of the technique beyond the context of scientific research.

Trace element concentrations in the moss Hypnum cupressiforme growing in a presumably unpolluted area.

In this study we determined the concentrations of As, Cd, Hg, Ni and Pb in samples of the moss Hypnum cupressiforme collected during 5 different sampling surveys (2006-2014) in a presumably unpolluted area in northern Spain (25 sampling sites). We then applied factor analysis (FA) to the data to explore the factors underlying the spatial and temporal variability in the concentrations. The percentage of variance explained by the FA ranged between 34 and 98%, and was usually higher than 70%. The FA yielded 5 factors that explained the variance in the concentrations of Cd, As, Hg and Pb in all sampling surveys and also a single factor that explained the variance in Hg and Pb concentrations in 2006. Although the lack of obvious sources of pollution in the study region (at least for the elements considered) suggests that most elements (except perhaps Ni) probably originated from long-range atmospheric transport, this would not explain the results of the FA. We suggest that rather than being due to the origin of the pollutants (as frequently assumed), the spatio-temporal variability in the concentrations of these elements is probably determined by a series of other factors: the physicochemical characteristics of the pollutants and of the moss binding surfaces, physiological processes (e.g. moss growth), and the characteristics of the sampling sites (e.g. vegetation cover, elevation, slope, aspect). We therefore conclude that the assumption that variations in element concentrations in moss tissues are due to the origin of the pollutants is an oversimplification that leads to erroneous interpretation of the results of biomonitoring studies with terrestrial mosses.

A critical review of protocols for moss biomonitoring of atmospheric deposition: sampling and sample preparation.

Currently, the most important guideline for the application of the moss technique to monitor the atmospheric deposition of heavy metals is the "Heavy metals, nitrogen and POPs in European mosses: 2015 survey" published by the UNECE ICP Vegetation. Two main problems have been identified with this guideline: i) some of the recommendations regarding the methodological aspects involved in the application of the moss technique are not based on scientific criteria; and, ii) some recommendations in the manual are very vague and some aspects are even left out (e.g., elevation, distance to the coast). As a result there exists a high variability in the application of the protocol and many scientists adapt it to the specific conditions in the studied areas without evaluating how changes affect the results obtained. Therefore, in this article a total of 369 studies were reviewed including both methodological and application studies of the passive biomonitoring of the atmospheric deposition of heavy metals with terrestrial mosses. The results of this review have shown on the one hand, that none of the articles completely accomplished the ICP-Vegetation protocol suggestions, either because the information regarding some aspects was lacking or simply because the authors did not follow the manual suggestions. On the other hand, it was found that the results of methodological studies sometimes contradicted the ICP Vegetation manual recommendations. Thus, a new protocol in which each suggestion has been carefully and rigorously contrasted with the available literature has been proposed in this paper. In addition, practical and economic issues have also been considered and much more concise suggestions have been proposed which would facilitate its fulfilment in a more objective way.

Relationship between trace metal concentrations in the terrestrial moss Pseudoscleropodium purum and in bulk deposition.

The relationship between trace metal concentrations in Pseudoscleropodium purum (Hedw.) M. Fleish. and bulk deposition (BD) was examined at 21 sites (SS) to ascertain whether the lack of correlations reported in previous studies are due to limitations in the experimental design. Thus, we implemented some improvements to it and examined the correlations in depth. We could conclude that this relationship is determined by other variables rather than by the pollutant inputs themselves (BD). Thus, whether or not moss concentrations and bulk deposition are significantly correlated will depend on the combination of the above referred factors in a given area at a given time. Therefore, P. purum in particular, and most likely native terrestrial mosses in general, are less than ideal biomonitors for yielding absolute values of atmospheric trace metal deposition. We recommend their use as qualitative indicators and the use of classical methods to obtain absolute values.

Spatial structure of trace elements in extensive biomonitoring surveys with terrestrial mosses.

The size of the area affected by contamination processes mainly depends on the type of contaminant and the way it is emitted. In regular sampling, the size of the sampling grid will therefore interact with the scale at which the contamination processes are produced, for each contaminant, so that the grid will only enable characterization of those processes that occur at scales larger than the grid, i.e. large scale processes. The present study analysed the data corresponding to tissue concentrations of Cd, Cu, Fe, Hg, Mn, Pb, Se, V and Zn in the terrestrial moss Pseudoscleropodium purum obtained in regular sampling surveys with grids of different sizes (15x15, 7.5x7.5 km) plus a series of accessory points located at 1 km from the 15x15 km sampling grid; all sampling sites were located in Galicia and were sampled in March 2004. The objective of the study was to characterize the interaction between the scale of the sampling grid and the scale at which the contamination processes are produced, for each contaminant, to enable adjustment of the size of the grids to the scale at which the contamination processes are mainly produced. For this purpose, the spatial structure of the concentration of each element was analysed by use of semivariograms created with a robust estimator of the semivariance. The results of the study verified the existence of a real interaction between the scale of the sampling grid and the scale of the contamination processes. The results also demonstrated how in the study area, the contamination processes associated with Cd, Pb and V are generally small scale processes, whereas those associated with Cu, Hg and Se are generally large scale processes.

Characterization of Neisseria polysacchareae sp. nov. (Riou, 1983) in previously identified noncapsular strains of Neisseria meningitidis.

A study of 216 noncapsular strains of Neisseria meningitidis isolated from patients and carriers received in the Meningococcus Reference Laboratory between 1978 and 1984 is reported. The characterization of the strains consisted of biochemical tests for the following characteristics used for the differentiation of Neisseria species: oxidase, catalase, and beta-galactosidase activities; sugar degradation; nitrate and nitrite reduction; DNase activity; polysaccharide production with 5% sucrose; aminopeptidase activity; and growth in Thayer-Martin and Catlin media. Of the strains studied, 50 showed characteristics of a new taxon recently described (Neisseria polysacchareae). Characteristics that differentiated these strains from meningococcal isolates were polysaccharide production with 5% sucrose, gamma-glutamylaminopeptidase activity, and a requirement for cysteine or cystine for growth in Catlin medium. All of the N. polysacchareae strains identified were isolated from the nasopharynx of healthy carriers.