What potential do mosses have as biomonitors of POPs? A comparative study of hexachlorocyclohexane sorption.

Persistent organic pollutants (POPs) pose a significant global threat to human health and the environment, and require continuous monitoring due to their ability to migrate long distances. Active biomonitoring using cloned mosses is an inexpensive but underexplored method to assess POPs, mainly due to the poor understanding of the loading mechanisms of these pollutants in mosses. In this work, Fontinalis antipyretica (aquatic moss) and Sphagnum palustre (terrestrial moss) were evaluated as potential biomonitors of hexachlorocyclohexanes (HCHs: α-, ß-, γ-, δ-HCH), crucial POPs. Moss clones, grown in photobioreactors and subsequently oven-dried, were used. Their lipid composition and distribution were characterized through molecular and histochemical studies. Adsorption experiments were carried out in the aqueous phase using the repeated additions method and in the gas phase using an active air sampling technique based on solid-phase extraction, a pioneering approach in moss research. F. antipyretica exhibited greater lipid content in the walls of most cells and higher adsorption capacity for all HCH isomers in both gaseous and liquid environments. These findings highlight the need for further investigation of POP loading mechanisms in mosses and open the door to explore other species based on their lipid content.

Unravelling the metal uptake process in mosses: Comparison of aquatic and terrestrial species as air pollution biomonitors.

Transplanted mosses have been widely shown to be excellent tools for biomonitoring air pollution; however, it is not clear how the functional groups present on their surfaces affect the uptake of metal cations. In the present study, we examined differences in trace metal accumulation in two terrestrial and one aquatic moss species, and investigated whether the differences depended on their physico-chemical characteristics. In the laboratory, we determined C, N and H contents in their tissues and obtained the ATR-FTIR spectra (to identify the presence of functional groups). We also conducted surface acid-base titrations and metal adsorption assays with Cd, Cu and Pb. In the field, we exposed transplants of each species near different air-polluting industries, and determined the mosses enrichment of Al, Cd, Co, Cr, Cu, Fe, Ni, Pb and V. Laboratory results demonstrated higher metal uptake capacity in the terrestrial mosses Sphagnum palustre and Pseudoscleropodium purum, compared to that in the aquatic moss Fontinalis antipyretica, which can be attributed to a greater abundance of acidic functional groups (i.e. negatively charged binding sites) on the surface of the terrestrial mosses. The affinity of moss for certain elements depends on the abundance and nature of surface functional groups. Accordingly, the metal concentrations generally reached higher levels in S. palustre transplants compared to the other species, except for the uptake of Hg, which was higher in F. antipyretica. However, the findings also suggest an interaction between the type of environment (terrestrial or aquatic) and the moss characteristics that may influence the abovementioned trend. Thus, irrespective of the physico-chemical characteristics, metal uptake varied depending on the environment of origin of the mosses "i.e. atmospheric or aquatic". In other words, the findings suggest that species that accumulate more metals in terrestrial environments will accumulate lower amounts of metals in aquatic environments and vice versa.

Global decrease in heavy metal concentrations in brown algae in the last 90 years.

In the current scenario of global change, heavy metal pollution is of major concern because of its associated toxic effects and the persistence of these pollutants in the environment. This study is the first to evaluate the changes in heavy metal concentrations worldwide in brown algae over the last 90 years (>15,700 data across the globe reported from 1933 to 2020). The study findings revealed significant decreases in the concentrations of Cd, Co, Cr, Cu, Fe, Hg, Mn, Pb and Zn of around 60-84% (ca. 2% annual) in brown algae tissues. The decreases were consistent across the different families considered (Dictyotaceae, Fucaceae, Laminariaceae, Sargassaceae and Others), and began between 1970 and 1990. In addition, strong relationships between these trends and pH, SST and heat content were detected. Although the observed metal declines could be partially explained by these strong correlations, or by adaptions in the algae, other evidences suggest an actual reduction in metal concentrations in oceans because of the implementation of environmental policies. In any case, this study shows a reduction in metal concentrations in brown algae over the last 50 years, which is important in itself, as brown algae form the basis of many marine food webs and are therefore potential distributors of pollutants.

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.

Can proteomics contribute to biomonitoring of aquatic pollution? A critical review.

Aquatic pollution is one of the greatest environmental problems, and therefore its control represents one of the major challenges in this century. In recent years, proteomics has emerged as a powerful tool for searching protein biomarkers in the field of pollution biomonitoring. For biomonitoring marine contamination, there is a consensus that bivalves are preferred organisms to assess organic and inorganic pollutants. Thus, the bivalve proteome was intensively studied, particularly the mussel. It is well documented that heavy metal pollution and organic chemicals altered the structural proteins causing degradation of tissues of molluscs. Also, it is well known that proteins involved in stress oxidative such as glutathione and enzymes as catalase, superoxide dismutase or peroxisomes are overexpressed in response to contaminants. Additionally, using bivalves, other groups of proteins proposed as pollution biomarkers are the metabolic proteins. Even though other marine species are used to monitor the pollution, the presence of proteomic tools in these studies is scarce. Concerning freshwater pollution field, a great variety of animal species (fish and crustaceans) are used as biomonitors in proteomics studies compared to plants that are scarcely analysed. In fish species, proteins involved in stress oxidative such as heat shock family or proteins from lipid and carbohydrate metabolism were proposed as candidate biomarkers. On the contrary, for crustaceans there is a lack of proteomic studies individually assessing the contaminants. Novel scenarios, including emerging contaminants and new threats, will require proteomic technology for a systematic search of protein biomarkers and a greater knowledge at molecular level of those cellular pathways induced by contamination.

Optimal number of Fucus vesiculosus samples to differentiate between sites affected by distinct levels of heavy metal contamination.

The presence of trace elements in marine habitats is a serious environmental problem which increasingly affects ecosystem and human health. The use of macroalgae as contamination biomonitors represents a valuable alternative approach to traditional physicochemical methods. The present study was carried out to determine the optimal number of samples of Fucus vesiculosus needed to detect statistically significant differences in the mean concentrations of Al, As, Cd, Co Cr, Cu, Fe, Hg, Ni, Pb, Zn, N and δ15N between two sampling sites affected by different levels of contamination. For this purpose, we plotted the density distributions of the concentrations of the different elements and examined the local variability at three sites. For sites with mean concentrations differing by more than 30 %, 20 samples were sufficient to detect significant differences for all of the elements, except Cr. The proposed methodology could be used in other studies in the absence of specific research on each species and region.

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.

Testing a novel biotechnological passive sampler for monitoring atmospheric PAH pollution.

In this study we evaluated a new type of passive air sampler, the "mossphere" device, filled with a Sphagnum palustre clone. For this purpose, we compared the atmospheric levels of polyaromatic hydrocarbons (PAHs) collected using this device and those collected in conventional bulk deposition and particulate matter (PM10) samplers. All three types of samplers were exposed at 10 sites affected by different levels of pollution and located in two different climate zones. The bulk deposition/ mossphere comparison yielded a greater number of significant regressions with higher coefficients of determination than the PM10/ mossphere comparison. No significant regressions were observed for 3-ring PAHs in either comparison. The mosspheres explain ca. 50% of the variability of the concentrations of 4-, 5- and 6-ring PAHs and total PAHs detected in PM10 and ca. 70% of the corresponding concentrations detected in the bulk deposition. The use of the Sphagnum clone enables standardization of the set-up, thus making the mossphere device a good sampling tool for monitoring 4-, 5- and 6-ring and total PAHs, especially those associated with bulk deposition. The findings indicate the potential usefulness of this innovative technology for mapping PAH levels.

Methodological advances to biomonitor water quality with transplanted aquatic mosses.

The active biomonitoring technique has been demonstrated to be an excellent tool for monitoring water quality; however, further improvement of the protocol is urgently needed. The present study was carried out to determine the best options for various methodological aspects of monitoring some metals and metalloids (i.e. Al, As, Cd, Co, Cu, Fe, Hg, Ni, Zn and Pb): i) the type of transplant, ii) pre-exposure washing (with or without cellular extractants), iii) the ratio between moss weight and bag surface area, and iv) the depth at which the bags are exposed. The importance of the different methodological aspects in the outcome of biomonitoring studies was also assessed by considering the results of the present and other previously published studies. Regarding the type of transplant, the traditionally used net bags were the best option for enclosing the moss; in addition, washing the moss with extracellular extractants (i.e. EDTA) prior to exposure increased the sensitivity of the technique and reduced the required exposure time (i.e. one week). For the amount of moss packed in each bag, a ratio of 12.5 mg cm-2 was the best choice. Finally, the depth at which the transplants were exposed did not affect pollutant accumulation (in shallow rivers, reservoirs or dams). Pollutant concentrations were also not affected by the existence of thermocline in deep waters during warmer months. Different methodological aspects involved in applying this technique determine the final concentrations of metals in moss. Although the influence of those was variable, for most elements (i.e. As, Cd, Co, Cu, Hg, Pb, Zn) 80% of the total variance was explained by 3-4 aspects, being species selection, devitalization treatment, duration of exposure, and number of transplants exposed the most important.

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.

Biomonitoring freshwater FISH farms by measuring nitrogen concentrations and the δ15N signal in living and devitalized moss transplants.

The trophic balance of freshwater aquaculture activities has traditionally been monitored by chemical analysis of water; however, the parameters measured are usually characterized by high temporal variability. Aquatic mosses can be used as biomonitors as they integrate both continuous and episodic contamination events. Here we report, for the first time, a method for monitoring N enrichment in the surroundings of fish farms by measuring the N content and isotopic signal (δ15N) of transplanted living and devitalized specimens of the aquatic moss Fontinalis antipyretica. For this purpose, moss samples ("moss bags") were exposed at increasing distances (10, 100, 300 and 1000 m) up- and downstream of the effluent discharge points of four trout farms, for 10 and 30 days. The low natural (background) variability in δ15N in upstream samples enabled detection of outlier values, caused by aquaculture discharges, at distances of 10 and 100 m downstream, especially in devitalized moss and after 10 days of exposure. However, the unexpectedly low N contents of moss samples exposed close to the discharge points complicates interpretation of the high levels of N forms detected by conventional physicochemical analysis of water. Although the mechanisms that modify N parameters in moss tissues were not clear, measurement of the isotopic signal δ15N in devitalized moss exposed for 10 days proved useful for monitoring the N pollution associated with intensive freshwater aquaculture.

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.

Sphagnum palustre clone vs native Pseudoscleropodium purum: A first trial in the field to validate the future of the moss bag technique.

Although a large body of literature exists on the use of transplanted mosses for biomonitoring of air pollution, no article has addressed so far the use and the accumulation performance of a cloned moss for this purpose. In this work, a direct comparison of metal accumulation between bags filled with a Sphagnum palustre L. clone or with native Pseudoscleropodium purum Hedw., one of the most used moss species in biomonitoring surveys, was investigated. The test was performed in sites with different atmospheric contamination levels selected in urban, industrial, agricultural and background areas of Italy and Spain. Among the eighteen elements investigated, S. palustre was significantly enriched in 10 elements (Al, Ba, Cr, Cu, Fe, Hg, Pb, Sr, V and Zn), while P. purum was enriched only in 6 elements (Al, Ba, Cu, Hg, Pb and Sr), and had a consistently lower uptake capacity than S. palustre. The clone proved to be more sensitive in terms of metal uptake and showed a better performance as a bioaccumulator, providing a higher accumulation signal and allowing a finer distinction among the different land uses and levels of pollution. The excellent uptake performance of the S. palustre clone compared to the native P. purum and its low and stable baseline elemental content, evidenced in this work, are key features for the improvement of the moss bag approach and its large scale application.

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.

PAH detection in Quercus robur leaves and Pinus pinaster needles: A fast method for biomonitoring purpose.

Due to the complexity and heterogeneity of plant matrices, new procedure should be standardized for each single biomonitor. Thus, here is described a matrix solid-phase dispersion extraction method, previously used for moss samples, improved and modified for the analyses of PAHs in Quercus robur leaves and Pinus pinaster needles, species widely used in biomonitoring studies across Europe. The improvements compared to the previous procedure are the use of Florisil added with further clean-up sorbents, 10% deactivated silica for pine needles and PSA for oak leaves, being these matrices rich in interfering compounds, as shown by the gas chromatography-mass spectrometry analyses acquired in full scan mode. Good trueness, with values in the range 90-120% for the most of compounds, high precision (intermediate precision between 2% and 12%) and good sensitivity using only 250mg of samples (limits of quantification lower than 3 and 1.5ngg(-1), respectively for pine and oak) were achieved by the selected procedures. These methods proved to be reliable for PAH analyses and, having advantage of fastness, can be used in biomonitoring studies of PAH air contamination.

Evaluation of the use of moss transplants (Pseudoscleropodium purum) for biomonitoring different forms of air pollutant nitrogen compounds.

We investigated whether three different types of moss transplants (devitalized moss bags with and without cover and auto-irrigated moss transplants) are suitable for use as biomonitors of the deposition of oxidised and/or reduced forms of N. For this purpose, we determined whether the concentration of atmospheric NO2 was related to the % N, δ(15)N and the activity of the enzyme biomarkers phosphomonoesterase (PME) and nitrate reductase (NR) in the tissues of moss transplants. We exposed the transplants in 5 different environments of Galicia (NW Spain) and Cataluña (NE Spain): industrial environments, urban and periurban environments, the surroundings of a cattle farm and in a monitoring site included in the sampling network of the European Monitoring Programme. The results showed that the moss in the auto-irrigated transplants was able of incorporating the N in its tissues because it was metabolically active, whereas in devitalized moss bags transplants, moss simply intercepts physically the N compounds that reached it in particulate or gaseous form. In addition, this devitalization could limit the capacity of moss to capture gaseous compounds (i.e. reduced N) and to reduce the oxidised compounds that reach the specimens. These findings indicate that devitalized moss transplants cannot be used to monitor either oxidised or reduced N compounds, whereas transplants of metabolically active moss can be used for this purpose. Finally, the NR and PME biomarkers should be used with caution because of the high variability in their activities and the limits of quantification should be evaluated in each case.

Study of temporal trends in mercury concentrations in the primary flight feathers of Strix aluco.

Temporal trends in Hg concentrations were determined in the primary flight feathers of 146 specimens of Strix aluco which had died in various Wildlife Recovery Centres in Galicia (NW Spain) between 1997 and 2014. The aim of the study was to determine whether standardization of a primary flight feather (or feathers) in this species is essential for identifying temporal trends in Hg concentrations. For this purpose, we had to first standardize the feather(s) analyzed to enable comparison of the levels of Hg detected in different feathers. The results show a high degree of both inter and intra-individual variability but despite that, it was possible to identify P5 as the most representative feather taking into account the amount of metal excreted in each feather and the intra-individual variability: its median was 133ng, which represents 15% (from 7% to 15%) of the total Hg present in all the primary feathers. However, this "standard feather" did not reveal any temporal trend in Hg concentrations for the study period. This lack of trend was found irrespective of the feather considered and it is expected that detection of any existing trend would also not depend on the feather considered. We conclude that use of any particular feather is not essential for identifying temporal trends in Hg concentrations, because the pattern will be identified regardless of the feather selected.