Monitoring river water quality with transplanted ...

Ecological Indicators 81 (2017) 461–470

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Review

Monitoring river water quality with transplanted bryophytes: A methodological review S. Debéna, J.R. Aboala, A. Carballeiraa, M. Cesab, J.A. Fernándeza, a b

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Ecology Unit, Department of Functional Biology, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain University of Trieste, Department of Life Sciences, University of Trieste, I-34127 Trieste, Italy

A R T I C L E I N F O

A B S T R A C T

Keywords: Bryomonitoring Heavy metals Freshwater Water pollution

The aim of this literature review, which considers 47 articles published between 1989 and 2015, is to ascertain the current status of the active biomonitoring technique for assessing water quality and to evaluate the degree to which different aspects of the method have been standardized. Use of the tool is largely limited to Europe (83% of the articles reviewed). The technique has been used to biomonitor inorganic contaminants (in 96% of the studies) and, to a lesser extent, organic contaminants (4% of the studies). Only 25% of the articles concern methodological aspects of the technique. Moreover, most authors (78%) have only published one article on the topic, and many different protocols have been used in the various studies. As a result, the technique is not standardized, which hampers comparison of the results of different studies. We propose a protocol that would facilitate use of the technique for routine monitoring of the quality of river waters.

1. Introduction River waters (mainly those associated with urban and industrial areas) are affected by different anthropogenic activities that could lead to deterioration in water quality (e.g. increasing levels of pollutants or acidity) and also in the ecological status. There is also a growing demand for good quality water to be available for specified uses (e.g. as drinking water, and in agriculture). In response to this demand, different authorities have developed specific legislative measures to protect inland freshwaters (e.g. European Water Framework: Directive 2000/60/CE). Nowadays, the main tool used to assess water contamination is chemical monitoring, which provides information about the levels of different pollutants in the water column (e.g. heavy metals and organic compounds). Nevertheless, the data obtained in this approach reflect the concentration of pollutants at the time of sampling, but not episodic or intermittent pollution events (Greenwood and Roig, 2006). To resolve this problem, various biological matrices (e.g. algae, bryophytes, fishes, molluscs and macro-invertebrates) have been used to assess water quality. Bryophytes show important advantages relative to the other options for the following reasons: (i) pollutant uptake by bryophytes is mainly passive and scarcely affected by biotic factors; (ii) bryophytes usually have long live-cycles; (iii) they are easily sampled, identified and transplanted; (iv) they are resistant to water pollution and adverse environmental conditions; (v) they do not need to be feed;

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Corresponding author. E-mail address: [email protected] (J.A. Fernández).

http://dx.doi.org/10.1016/j.ecolind.2017.06.014 Received 7 September 2016; Received in revised form 15 March 2017; Accepted 9 June 2017 1470-160X/ © 2017 Elsevier Ltd. All rights reserved.

and (vi) they are non-invasive species. Using bryophytes to biomonitor water quality (also called ‘bryomonitoring’) also enables the simultaneous monitoring of a large number of compounds (i.e. organic, inorganic and even radioactive compounds) by analysis of a single sample as well as evaluation of water quality at small (e.g. around pollutant sources) and large scales (e.g. regional). In this respect, two types of bryomonitoring are clearly differentiated: (i) passive, using specimens growing naturally in an area (for reviews, see Whitton, 2003; Gecheva and Yurukova, 2014; Debén et al., 2015); and (ii) active, by transplanting plants from other locations. For active bryomonitoring, samples are collected from relatively unpolluted habitats and are then cleaned, selected and pre-treated before being exposed in a different environment. The use of transplanted bryophytes resolves various problems associated with the use of native specimens. Thus, active bryomonitoring can be used in sites where native bryophytes are scarce or absent. It also eliminates possible phenotypic or genotypic adaptation of native plants to contaminants in polluted areas. In addition, its improves the temporal interpretation of results because the duration of the exposure period is known (Debén et al., 2015). Finally, it is also possible to assess the magnitude of the pollution because the initial concentrations of elements in the transplants are known. However, until now the use of this tool has been restricted to scientific research, and it has not been officially used by environmental authorities to assess the level of pollution in river waters. One of the main causes of the limited use of the


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Most of the studies (96%) measured the concentrations of inorganic contaminants and only a small proportion measured organic contaminants (4%). The types of elements most frequently analysed are heavy metals (Zn, Pb and Cu, in more than 70% of the studies), metalloids and some nutrients (Fig. 4). Some anionic elements (such as P and S; e.g. Yurukova and Gecheva, 2003), persistent organic pollutants (such as PAHs and PCBs; e.g. Roy et al., 1996) and even some radioactive isotopes (such as Cs137; e.g. Hongve et al., 2002) have also been considered. The present review considers four key aspects in relation to standardization of the technique used for active biomonitoring of water quality with bryophytes: (i) selection and preparation of the bryophytes; (ii) preparation of the transplants; (iii) exposure of the transplants; and (iv) post-exposure treatments. As well as reviewing and discussing the literature consulted, we also consider whether each different stage of the process can be standardized or further research is required.

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Fig. 1. Number of articles published by each author on active biomonitoring of the quality of river waters with aquatic bryophytes.

technique is the lack of a standardized method and a well-defined protocol. In this review, after describing the current status of the methodology, we will assess the degree of standardization of each of the steps involved in active bryomonitoring of river water and we will identify the aspects that require further research. The final aim is to propose a protocol that would enable the routine use of bryophyte transplants for assessing water quality in river ecosystems.

3. Selection and preparation of the bryophytes Use of a standardized sample preparation procedure will ensure that comparable results are obtained. For this purpose, the following aspects must be taken into account: (i) selection of the species used as the biomonitoring agent; (ii) sample collection; (iii) selection of the material for transplants and (iv) pre-exposure treatments.

2. Current trends in the active biomonitoring of river water In this review paper, we provide a critical evaluation of the methods used in 47 articles concerning the active biomonitoring of river waters. We selected articles that quantified the concentrations of contaminants in transplanted bryophytes, as well as those involving experiments related to methodological aspects. The articles were all published between 1989 and 2015 and were located using the SciVerse SCOPUS online tool (www.scopus.com). The review highlights the low degree of standardization of the technique. Most authors (78%) have only published one article on the topic (Fig. 1), and many of the protocols have been used on only one occasion or by only one research group. However, the main aim of most of the studies considered (79%) was to biomonitor contamination (Fig. 2), and studies aimed at establishing a standardized method by investigating particular aspects of the method were much less abundant (22%). In fact, only 12% of the authors have published more than two articles on the topic and only 5 of these authors have investigated some aspect of the methodology (i.e. López et al., 1994; Mersch and Reichard, 1998; Vázquez et al., 2000b; Martins et al., 2010; Cesa et al., 2011, 2015). The huge variability in methods hampers comparison of the results obtained in different studies and sometimes restricts the conclusions that can be reached. Finally, use of the technique is mainly limited to Europe (80% of the articles reviewed, see Fig. 3).

3.1. Selection of species used as biomonitoring agent This is one of the key aspects that must be considered to enable valid comparison of the information obtained at different sampling stations (SS). After grouping the diverse synonyms reported in the literature, in accordance with Hill et al. (2006), we found that 11 species have been used and that 55% of these have only been used on one occasion. Most of the bryophytes used are mosses, although liverworts have also been used (e.g. Engleman and McDiffett, 1996; Thiébaut et al., 2008). The frequency with which species of aquatic bryophytes have been used worldwide is summarised in Fig. 3. The moss Fontinalis antipyretica Hewd. has been used in 55% of the studies reviewed and Platyhypnidium riparioides (Hewd.) Dixon in 25% of the studies (note that according to Hill et al. (2006), Rhynchostegium riparioides (Hedw.) Cardot is a synonym of this species). These species are probably used because they are widely distributed, relatively large and easy to identify and handle (Martinez-Abaigar et al., 2002). The third most frequently used species is Hygrohypnum ochraceum (Turner ex Wilson) Loeske (in 9% of the studies). When different species are used in the same study, interspecific differences in the uptake capacity must be taken into account on Fig. 2. Number of articles published in different years on the use of transplanted aquatic bryophytes to biomonitor water quality. White bars: number of articles concerning methodological aspects; grey bars: number of articles involving use of the technique to monitor contamination.

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Fig. 3. Representation of the different bryophytes used as active biomonitors of river water quality in each continent and worldwide. The size of the circle indicates the frequency (number of articles) of use of the biomonitoring technique.

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Fig. 4. Elements most frequently determined in aquatic bryophytes for monitoring water quality.

Alkali and Alkali Eath metals 40 20 0 Zn Cu Pb Cd Ni Fe Cr Al Mn As Co Ca Hg K Mg Na V Sb Se Ag Ba Mo Pd Pt Sn Ti Elements measured

Comparison of the results obtained with different species is therefore not valid and selection of a species that can be used as a standard biomonitoring agent is required. The species selected should fulfil the following criteria (Ares et al., 2012): (i) it should be widely distributed; (ii) it should have structural or physico-chemical characteristics that enable efficient adsorption of contaminants from water, and (iii) it should be one of the most commonly used and about which most information is available. In addition, and perhaps most importantly, the species selected should be able to be cultured under laboratory conditions to yield large amounts of biomass. Both F. antipyretica and P. riparioides are widely distributed in the northern hemisphere. Although the latter displays a higher capacity to accumulate contaminants, the former species is more commonly used in active biomonitoring studies. It is therefore difficult to establish which of these species should be used as a standard biomonitoring agent. No studies have yet been carried out to compare the growth rates of these species under laboratory conditions. This type of information would help researchers choose between these species.

comparing the results obtained. However, such differences have rarely been investigated in relation to moss transplants. Mersch and Reichard (1998) studied interspecific differences in metal accumulation (i.e. Cu, Cr, Ni, Pb and Zn) at a series of SS by comparing transplants of the aquatic bryophytes P. riparioides, F. antipyretica and Cinclidotus danubicus Schiffner & Baumgartner. These authors found that P. riparioides accumulated the highest concentrations of metals and C. danubicus the lowest concentrations. Cesa et al. (2015) compared transplants of the same species and reported similar findings: P. riparioides performed best in terms of uptake of the elements considered (i.e. As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn); C. aquaticus was the weakest accumulator, and F. antipyretica showed an intermediate efficiency. Thiébaut et al. (2008) also found that accumulation of Al and Fe was affected by acidic conditions: transplanted specimens of P. riparioides accumulated the highest relative amount of Al and the lowest amount of Fe (p < 0.05), whereas the acid-tolerant bryophyte Scapania undulata (L.) Dumort. accumulated more Fe and less Al. We have also found interspecific differences in biomonitoring studies with native mosses (Debén et al., 2015). 463


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the material can be separated when the transplants are prepared or after exposure. If devitalized moss is used, the apical segments must be separated when the material is cleaned (see Section 3.4.1), before the devitalization process and thus before exposure of the transplants.

3.2. Sample collection Another important aspect to consider when choosing which species to use for aquatic biomonitoring is the availability of unpolluted sites from which material can be collected. Such sites should comply with the following criteria: (i) they should be accessible; (ii) the selected species should be present at the site in sufficient quantities to enable collection without threatening conservation of the native population; (iii) the sites should be located upstream of any sources of anthropogenic pollution, and (iv) the concentrations of the different elements under consideration should be as low as possible. Even if all of these requirements were fulfilled, the availability and natural variability in the elemental composition of the moss may vary depending on natural and anthropogenic factors. One possible solution to this problem is to culture aquatic mosses, or a clone of a particular species, in the laboratory. The main advantage of using clones is the ready availability of high quality material for transplanting: the variability in the results would be minimal as the biological material would be cultured under the same conditions and would initially contain the same concentrations of elements (Rausch de Traubenberg and Ah-Peng, 2004). The clone of F. antipyretica produced by these authors has been lost. However, we know that at least two axenic cultures of moss clones have been established: one of P. riparioides (Ralf Reski pers. comm.) and the other of F. antipyretica, grown in our laboratory. The use of cultured moss for biomonitoring studies would prevent damage to the environment caused by frequent collection of samples and would ensure a constant supply of the material required. We have therefore considered the rate of growth of moss species under laboratory conditions (i.e. in bioreactors) as one of the fundamental aspects for selecting the species used as a biomonitoring agent (see Section 3.1).

3.4. Pre-exposure treatments Once the material has been selected, it may or may not be treated before exposure in the study site; treatments are usually restricted to cleaning the material (e.g. in situ, by hand with water in the laboratory, etc.). Mosses can also be devitalized before exposure. 3.4.1. Sample cleaning method In most of the articles consulted (42%), the samples were washed in the river water at the SS to remove sediment remains, epifauna and adhered particles (in situ cleaning). In some studies (3%), the moss specimens were strained to remove excess water (Mersch and Reichard, 1998). Once in the laboratory, the samples can be cleaned in the same way by manual removal of adhered material, although the most usual method is to wash the samples with water. In 68% of the articles reviewed, no information was provided about the cleaning method used (e.g. Rasmussen and Andersen, 1999; Samecka-Cymerman et al., 2005; Rabnecz et al., 2008), and in the other 32% of the articles, the samples were washed with water. Washing with water usually involves a single wash (16% of the studies; e.g. Carter and Porter, 1997; Vázquez et al., 2000a,b) with either distilled water (in 5% of the studies; e.g. Thiébaut et al., 2008) or deionized water (in 13% of the studies; e.g. Herrmann et al., 2012). Shaking, which directly affects the efficacy of the washing process, was only used in one study (Herrmann et al., 2012), and no information about the intensity of shaking was provided. Moreover, no information was given in any of the studies about the ratio between the weight of the sample cleaned and the volume of water used. From experience, we know that aquatic moss is often covered with plant remains and silt, and that washing the samples is therefore essential. Moreover, we recommend using cellular extractants that reduce the initial concentrations and the associated variance while increasing the accumulation capacity of the moss. This has been tested with Sphagnum palustre cultured in the laboratory; washing the moss samples with EDTA decreased the coefficient of variation in the concentrations to below 10% for 11 of the 12 elements studied (Paola Adamo, pers. comm.), which represents an improvement in the technique. Although the efficiency of the washing procedure has not been investigated in aquatic bryophytes, standardization of this stage is necessary. We recommend cleaning the material in situ during sample collection and that the washing process should be as thorough as possible, to facilitate posterior cleaning. In the laboratory, manual cleaning of the material selected for making transplants should consist of manual removal of most epiphytes, plant remains and particles from the surface of the bryophytes plus a single wash in water (10 g d.w. of moss per 1 L distilled water with shaking for 20 min). As proposed by Ares et al. (2012) for washing terrestrial moss, we recommend that the washing stage also includes washing with cellular extractants (12 g dw of moss per 1 L EDTA (10 mM) with shaking for 20 min), followed by two washes in distilled water.

3.3. Selection of material for transplanting Appropriate selection of the part of the shoots used to prepare the transplants is essential for producing material that is as homogeneous and representative as possible for valid comparison of SS. Studies carried out with native mosses (Siebert et al., 1996 Martínez-Abaigar et al., 2002) have demonstrated significant differences in accumulation of some elements along the length of the shoot, i.e. from the base to the apex. Nonetheless, many of the studies reviewed (17%) do not provide any information about the part of the plant used. In most of the studies (63%) whole moss plants were used to prepare the transplants, and in many of the studies (43%), the apical segments (2–5 cm) or green parts were separated from the shoots after exposure. In 20% of the studies reviewed, apical segments (of between 2 and 4 cm) or green parts were used. In a recent study, Cesa et al. (2015) investigated differences in the concentrations and variability of As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in two different parts of the moss (whole plant and shoot tips) in three moss species (C. aquaticus, F. antipyretica and P. riparioides). These authors recommend that, for standardization of the moss-bag technique, shoot tips rather than whole plants should be analysed, as this (i) often reduces data variability, at least in transplants of C. aquaticus and F. antipyretica, (ii) increases element concentrations in P. riparioides, and (iii) enables comparison of data both in space (the same material transplanted to different survey areas) and in time (temporal trend assessment). The disadvantage of using apical segments (approx. 2–3 cm) is the potential difficulty in obtaining sufficient material (although this will not be a problem if cultured mosses are used as all of the material will comprise shoot tips). The green parts of shoots provide more material; however, no studies have compared apical and green parts, which a priori are physiologically homogeneous and may also accumulate contaminants at the same rate. The vital status of the material (see Section 3.4.2) to be transplanted may determine the time at which selection and particularly the green parts of shoots are separated. If live moss is used,

3.4.2. Vital status Devitalized moss was used to prepare the transplants in only three of the articles reviewed (Yurukova and Gecheva 2003, 2004; Cesa et al., 2011). However, some advances in standardizing the active biomonitoring of air quality have demonstrated the benefit of devitalizing the material, particularly by oven-drying, before exposure (Adamo et al., 2007). Devitalizing treatment can prevent interference from metabolic processes (Fernández et al., 2010), thus allowing more direct comparisons between the results of different surveys (Castello, 1996; Adamo et al., 2007, 2008; Giordano et al., 2009; Tretiach et al., 2007). Furthermore, this treatment may improve the applicability of the technique 464


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articles reviewed, and in the remaining articles, the size class distribution (mm2) was extremely variable: 1–5 (in 19% of articles), 6–30 (in 38%), 31–70 (in 15%), 71–100 (in 23%) and > 100 (in 4%). The only study in which different mesh size are compared was that carried out by Kelly et al. (1987), who concluded that muslin is not suitable for making the bags because the accumulation of Zn in mosses was greatly reduced by the fine mesh. These authors also compared nylon of two different mesh sizes (49 and 81 mm2) and although they conclude that these mesh sizes had little effect on Zn accumulation, they recommended using material of mesh size > 49 mm2. Other authors reported that material of mesh size 100 mm2 did not significantly interfere with light or nutrient reception (Mouvet,1984; López et al., 1994; Vázquez et al., 2000a,b). However, when apical segments and devitalized material are used, a smaller mesh size must be used in order to minimize loss of moss during the exposure period. Material of mesh size 16 mm2 has been used in the most recent studies (Cesa et al., 2010, 2011, 2013).

as it enables production of a ready supply of stock material that is easy to store. Cesa et al. (2011) demonstrated that pre-treatment by oven-drying at 105 °C does not substantially modify the uptake efficiency of several trace elements, especially of heavy metals, in the aquatic moss P. riparioides, and the uptake was sometimes even higher than in live moss. In a recent study (Debén et al., 2016), we compared different devitalizing treatments (i.e. oven-drying at 100 °C, oven-drying with a 50–80–100 °C temperature ramp, and boiling in water) applied to F. antipyretica under field conditions. We demonstrated that devitalizing treatments do not inhibit the capacity of moss to accumulate contaminants in the field and that devitalizing the material by oven drying with an increasing temperature ramp is more suitable than other methods, as it decreases the variability in the concentrations of elements. 4. Preparation of transplants Standardization of the following aspects of the protocol is essential: (i) the type of transplant used (e.g. moss bag, boulder with mosses attached, etc.) and the associated characteristics (for moss bags, the mesh size, mesh net material and the ratio between the amount of moss used and the size of the transplant) and (ii) the pre-exposure storage method.

4.1.2. Mesh net material As well as the mesh size, the type of material used (i.e. cotton, nylon, etc.) can also interfere in the absorption process and must be taken into consideration. However, in 16% of the articles reviewed, the composition of the material was not indicated. The most commonly used materials are nylon (34%) and plastic (47%). Glass fibre and nylon or other high density plastics are the most suitable types of material because they are inert and therefore do not affect the absorption process. Washing the mesh with dilute acid before preparing the transplants is recommended to eliminate any trace contaminants. This particularly applies to commercially available material such as mosquito netting, which may be impregnated with insecticide or insect repellent (Ares et al., 2012).

4.1. Type of transplant The first step in preparing transplants is to choose a type of support that will enable the bryophytes to be positioned in a realistic way. Different types of transplant-support systems are described in the literature. In the most basic system, used in only 13% of the studies reviewed, moss-covered boulders were collected from the reference site and transported to the monitoring site (Wehr et al., 1987; Sérgio et al., 2000); however, this is not a good option for long-term and/or large scale monitoring programmes because of the lack of sufficient quantities of suitably-sized moss-covered boulders. In a similar system, Hongve et al. (2002) tied the mosses together with a thin nylon rope to form bunches that they then attached to stones. In other studies (8% of the articles reviewed), the moss samples were placed inside open-ended or perforated plastic tubes or bottles through which water was able to pass (e.g. Mouvet, 1984; Rabnecz et al., 2008). Figueira and Ribeiro (2005) proposed a device consisting of a plastic tube with a longitudinal slit, into which only the basal part of the moss is placed, allowing the remainder of the plant to float free in the direction of the streamflow. However, the most common system (79% of the articles reviewed) is the moss bag technique, which consists of placing the moss samples in mesh bags. Kelly et al. (1987) reported that there were no significant differences (p < 0.05) in Zn, Cd and Pb accumulation in moss transplants (F. antipyretica and P. riparioides) attached to boulders or enclosed within mesh bags. Moreover, preparation of moss bags is simple and the materials used are inexpensive and readily available. The use of mesh bags is so far considered the best option for holding moss transplants. However, comparative studies of the different types of supports are required (with particular emphasis on the variability in results and considering various different contaminants). The characteristics of the bag can influence the results obtained and should therefore be considered for standardization of the method.

4.1.3. Amount of moss used and size of transplant One of the main aspects determining the amount of moss used in each transplant is the availability of sufficient material at the reference site. Sufficient quantities of moss should be able to be collected without threatening the conservation status of the native population. However, sufficient material must be collected to enable determination of both inorganic (heavy metals and metalloids) and organic contaminants (dioxins, furans, PAHs and PCBs) plus some material to be stored for future analysis. Finally, the mechanical effect of the water on the transplant system during exposure can lead to loss of large amounts of moss from the bags. We have found that the loss of material from bags (mesh size 4 mm2) containing devitalized F. antipyretica ranged between 20% after exposure for 5 days and 54% after exposure for 20 days (Debén et al., 2016) In the literature consulted, the amounts of moss used were very variable, ranging from 5 g f.w. (Beaugelin-Seiller et al., 1994) to 100 g f.w. (Samecka-Cymerman et al., 2005), although the amount most commonly used (in 21% of the articles reviewed) is 10–15 g f.w. The amount of material needed will depend on storage requirements and the analytical requirements for the type of contaminant, e.g. 0.2 g d.w. for metals, 0.25 g d.w. for PAHs (Concha-Graña et al., 2015) and 1 g d.w. for dioxins and furans. The amount generally varies between 1 and 5 g d.w. Selection of the most suitable size of bags is also very important as this determines the ratio between the weight of moss and the surface area of the bag. In turn, the ratio affects the efficiency of contaminant uptake by the transplanted moss. However, this ratio is not mentioned in any of the articles reviewed, and in most of the articles (81%) it cannot be calculated from the data provided. In those studies in which the ratio could be calculated (mg cm−2), it was fairly similar: < 28 mg cm−2 (in 29% of articles), 28–38 mg cm−2 (in 29%), 38–50 mg cm−2 (in 43%). In theory, using the minimum amount of moss possible to prevent the material being squashed will prevent differences in accumulation

4.1.1. Mesh size Selection of a suitable mesh size is a compromise between maximizing interception of contaminants and minimizing the risk of material being lost. On the one hand, fine mesh may enhance retention of some particles, thus affecting uptake of these by the moss. On the other hand, wide-spaced mesh may lead to loss of large amounts of moss being washed through the spaces. Despite its importance, the mesh size is not specified in 32% of the 465


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rates in the centre and close to the inner surface of the bags (Kelly et al., 1987). This has been demonstrated in terrestrial mosses (Ares et al., 2014). These authors found that the final concentrations of all elements studied (i.e. Cd, Cu Hg, Pb and Zn) generally decreased as the weight of Shpagnum denticulatum Brid. in the moss bags increased, and they established the optimal ratio between moss weight and bag surface area as 5.68 mg cm−2. Although this aspect has not yet been investigated in aquatic bryophytes, a lower ratio between the weight of moss and the surface of the bag can be used if the apical segments are separated before exposure of the transplants. Until further research allows a specific recommendation to be made, we therefore recommend using a ratio of 3.5 mg cm−2, which is similar to that recommended by Ares et al. (2014), although the loss of material during exposure of transplants in the aquatic environment may be greater than in the terrestrial environment.

These include the characteristics of the sampling site (e.g. depth and width of the river and type of riverbed) and practical aspects such as location of the moss bags as far as possible from obstacles that may interfere in the exposure and use of supports made from inert materials that are not affected by the contaminants under study and do not release contaminants during exposure. Although no studies have specifically investigated the exact location on the riverbed where the transplants should be placed, the depth should be sufficient to ensure that the moss is submerged throughout the exposure period. Stagnant water should be avoided, as should the edges of the river (as material from the riverbank may fall into the water and sweep the transplants away). Future research should address these aspects; however, in the meantime, we suggest that whenever possible the transplants should be attached to the riverbed, with plastic ties or concrete blocks, in the middle of the river (or as close to the middle as possible).

4.2. Pre-exposure storage

5.2. Duration of exposure

To ensure optimal conditions of live bryophytes, the time between collecting the material from the reference site and exposing it in the study sites should be minimal. In 16% of the articles reviewed, the authors recommend collecting the moss on the same day or on the day before the start of exposure (e.g. Mersch and Pihan, 1993; Cesa et al., 2013). However, this is often impossible, particularly when large amounts of material are used. In such cases, the moss samples can be stored for several days in an aquarium containing double distilled water, at a temperature close to that of the reference site, and with constant aeration. As the use of devitalized bryophytes is a recent development, no data are yet available on the possible effects of different types of storage on the transplants. Until this aspect is investigated, we suggest following the recommendations proposed for terrestrial bryophytes (Ares et al., 2012), i.e. to store the moss bags at low temperature (i.e. −20 °C) in individual sealed plastic bags (with e.g. a zip-lock system) to prevent loss of material, contamination of the samples or degradation by bacteria.

The quantities of elements accumulated by the mosses will largely depend on the duration of exposure. The process can be represented by a two-compartment model (water/moss) and first order kinetics (Claveri et al., 1994; Martins and Boaventura, 2002; Ferreira et al., 2009; Diaz et al., 2012). The metal content of the moss reaches saturation point when all of the ionic exchange sites are occupied. If the exposure time is very short, or the concentrations of the elements in the water are well below the saturation threshold, the uptake by the moss can be interpolated using a linear equation, as done for Hg and Sb in F. antipyretica (Diaz et al., 2012) and Hg in P. riparioides (Cesa et al., 2014). The exposure times usually range between 1 and 2 weeks (in 39% of the articles reviewed; e.g. Wehr et al., 1987; Nimmo et al., 2006; Herrmann et al., 2012) and 3–4 weeks (in 45% of the articles; e.g. Mersch and Johansson, 1993 Cenci, 2001; Cesa et al., 2013). Laboratory and field investigations have shown that the exchange kinetics are very rapid in mosses. Thus, according to Kelly et al. (1987), the exposure period should be at least 24 h, while López et al. (1994) recommended that the transplants should be exposed for at least 5 days. Cesa et al. (2009) reported that accumulation of Cu, Ni and Se in P. riparioides was maximal in the first two weeks, whereas accumulation of Mn continued to increase linearly over a period of four weeks. Similar uptake kinetics were also observed for Zn in P. riparioides (Wehr et al., 1987) and for Cr, Cu and Pb in F. antipyretica (Cenci, 2002). Fern & ndez et al. (2006) described the kinetics of both uptake and release, for Al, Co, Cu, Ni and Zn. These authors found that accumulation of metals in F. antipyretica is characterized by initial rapid accumulation in the extracellular compartments, followed by a gradual slowing down during intracellular accumulation of elements. When the contamination ceases, the release of contaminants occurs more slowly and is never complete. Extraction of the residual fraction of contaminants, which is probably located intracellularly (Mouvet and Claveri, 1999), enables detection of any sporadic contamination occurring several days before the transplants are collected. A poorly or non reversible enrichment process can also occur and involves deposition of Al, Fe and Mn oxides on the oldest parts of the gametophyte. Scanning electron microscopy studies revealed that these compounds occur as circular deposits distributed irregularly on the leaf surface (Sérgio et al., 1992, 2000). The gradual formation of these deposits is probably responsible for the increase in concentrations of the elements from the apical (youngest) parts to the basal (oldest) parts of the shoots observed in native moss (Wehr and Whitton, 1983; Wehr et al., 1983; Bruns et al., 1995; Siebert et al., 1996). In addition to the concentration of contaminants in the water, many chemical, physical and biotic factors (e.g. pH, solids in suspension, intercationic competition, etc.) can interfere in the response of bryophytes to contamination. The interactions between these factors and the exposure time will have different effects on the results obtained. Until

5. Exposure The prepared transplants are exposed to the environmental conditions in the study (monitoring) site. The final concentrations of the contaminants in the moss are affected by the combination of the different aspects of exposure (i.e. location of the transplants and type of support used, duration of exposure and number of bags per site). Furthermore, interpretation of the results will depend on whether or not some material is used to determine the initial concentrations. 5.1. Location of transplants and support In streams or rivers, the highly variable flow throughout the water column may affect the availability of contaminants and the uptake of these by moss. However, despite the importance of this aspect, 29% of the articles reviewed (e.g. Mersch et al., 1993; Sérgio et al., 2000) do not provide any information about how the transplants were arranged, their position in the water column or the method used to anchor them. The most common methods used to position the transplants are anchoring them to the riverbed with a boulder or concrete block (32%) (e.g. Mouvet, 1984 López et al., 1994) and suspending them midstream by attaching them to posts or stakes (11%) (e.g. Kelly et al., 1987; Cesa et al., 2006). Kelly et al. (1987) found no significant differences in Zn accumulation in transplants of P. ripariodes attached in these different ways, although they suggest that fixing the transplants to the substrate is the most realistic option. Moss transplants have also been suspended in the river from a line attached to a bridge (Yurukova and Gecheva, 2004) and held in the upper part of the stream attached to a float (Rabnecz et al., 2008). Different factors must be taken into account when selecting the method of location and the type of support used. 466


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sensitivity for detecting aquatic contamination. It is also important to take some measures to prevent contamination of samples outside of the exposure period or site. Handling of samples during collection after exposure of the transplants or their transport to the laboratory may lead to different levels of contamination or loss of adhered particles. To address this problem, control bags that are subjected to the same handling and transport stages, but are not located in the study sites, are sometimes included (e.g. Cesa et al., 2011; Herrmann et al., 2012). We recommend that the control bags should be hermetically sealed immediately after they are prepared and only opened when the exposed transplants are collected.

the effects of such factors are clarified, we recommend an exposure time of two weeks, as this may represent a good compromise between uptake efficiency and the risk of loss of moss bags due to accidents or adverse hydrological conditions. In any case, the exposure time may be modified depending on the specific characteristics of the sampling survey or economic conditions (e.g. for long-term studies may be interesting to use exposure times of four weeks). 5.3. Number of transplants per site The number of moss bags exposed in active biomonitoring studies ranges between 1 and 13. Although replicate bags were included in most studies (n = 2–3 in 23% of the studies, n = 4 in 8% of the studies, n = 5 in 8% of the studies and n = 6-13 in 15% of the studies), a substantial proportion (21%) of the studies did not include replicates. Herrmann et al. (2012) reported that the variability in Se uptake within five separate transplants of Hygrohypnum ochraceum was consistently low at all 14 SS considered, although they do not provide the corresponding data. Mersch and Reichard (1998) also observed a low degree of variability (coefficient of variation < 10%) in the concentrations of Co, Cr and Ni in transplants of P. ripariodes and C. danubicus. In the other studies that used replicate bags and those from which information can be extracted, the variability in the concentrations of elements in transplants of F. antipyretica was very low ( < 10%) for Co, Cr, Cu, Fe, Ni, V and Zn (Mersch and Reichard, 1998; Samecka-Cymerman et al., 2005; Rabnecz et al., 2008); intermediate (< 45%) for Al, Ba, Ca, Cd, Co, Cr, Cu, Fe, Mn, N, Ni, P, Pb, S and V (Samecka-Cymerman et al., 2005; Rabnecz et al., 2008); and extremely high in only a few cases (> 100%), for As, Cr, Cu and Pb (Rasmussen and Andersen, 1999; Rabnecz et al., 2008). One way of selecting the optimal number of moss bags would be to consider the level of error allowed in estimating the mean concentrations of contaminants in the moss tissues. However, given the lack of studies regarding the number of moss bags that should be located in each SS, we recommend the option most commonly used in previous studies when replicates bags were included, i.e. at least 3 bags per site.

6. Post-exposure treatments We recently established a protocol for cleaning, drying, homogenizing and storing aquatic bryophytes destined for used as passive biomonitoring agents (Debén et al., 2015). These treatments could also be used to process moss transplants after exposure at monitoring sites. We recommend restricting cleaning to the following: (i) in situ cleaning during sample collection (this should be as thorough as possible, to facilitate posterior cleaning); and (ii) manual cleaning (manual removal of most epiphytes, plant remains and particles from the surface of the bryophytes). Although in most of the articles reviewed (77%) the moss transplants were washed in the laboratory after exposure (once, with tap water in 15% of the studies and with distilled water in 21% of the studies), we do not recommend this procedure, as it alters the composition of the samples (Wells and Brown, 1990). In more than 70% of the studies reviewed, the samples were dried before being analysed. However, the temperature of drying varied from ambient (20 °C) to 105 °C, and the duration of drying varied between 4 h and 3 days (e.g. Wehr et al., 1987; Bruns et al., 1995; Cesa et al., 2011). The most common option consists of drying the samples at 40 °C for 2 or 3 days (in 26% of the studies). Following the previously established protocol, we propose that the moss should be dried at 40° C, to avoid loss of some elements (Lodenius et al., 2003), and that an aliquot of the sample should be dried at 100 °C (to constant weight), to determine the correction factor that should be applied to the element concentrations. Finally, although the samples were milled in only 11% of the studies reviewed, we recommend milling to homogenize the material and thus increase the analytical replicability. The ground moss samples should be stored in containers that will not affect the long-term stability of the material (e.g. glass, quartz or Teflon containers).

5.4. Initial concentrations and controls In most of the articles reviewed (61%), some of the material collected at the reference site is stored for subsequent determination of the initial concentrations of each of the contaminants considered. These values are then used to calculate the enrichment factor (EF = final concentration/initial concentration) and the net enrichment (NE = final concentration − initial concentration) for each of the transplants and contaminants studied. In some studies, the final concentrations in the transplants are compared with the concentrations in transplants located in the reference or other uncontaminated sites (e.g. Carter and Porter, 1997; Rabnecz et al., 2008). In others, they are compared with the concentrations of contaminants in the water or in native bryophytes in the study area (e.g. Carter and Porter, 1997; Deacon et al., 2001; Yurukova and Gecheva, 2004). However, within the same SS, there may be differences between transplanted and native bryophytes due to different exposure times, variations in concentrations of contaminants in the water prior to location of the transplants or to the possible development of tolerance to some elements by the native mosses. The variation in initial concentrations must also be taken into account. Couto et al. (2004) suggested testing the sensitivity of the mossbag technique by using the initial concentrations to calculate the limit of quantification (LOQ) of the technique (different from the analytical LOQ). This enables determination of the minimal concentration of a contaminant in the transplant that is considered different from the initial concentration. Use of a moss clone cultured under controlled conditions would provide much more homogeneous initial concentrations, thus decreasing the LOQ of the technique and increasing the

7. Evaluation of the methods used to date We assessed the consistency between the methods applied in the articles reviewed and the proposed recommendations (Table 1). Thus, the articles reviewed were qualified according to 12 aspects, and we awarded scores of 0 and 1 (for the vital status and the use of initial concentrations) or between 0 and 2 for the others aspects (where 0 indicates that no information is given about the aspect in question and 2 indicates maximal consistency with the recommendations). Thus, each article could be awarded a score of between 0 and 20 (Fig. 5). The results of the assessment revealed the wide variety of methods used and the general lack of attention given to developing the methodology. None of the studies reviewed fully complied with recommended procedures, and the overall score awarded was never higher than 15 (Fig. 5). In fact, 60% of the studies were awarded less than half of the maximum score. On the other hand, the frequent use of F. antipyretica and P. riparioides in moss bags indicates that the selection of the biomonitoring species and the type of transplant have been the aspects of the methodology most often considered.

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Table 1 Methodological aspects of the use of transplanted aquatic bryophytes to biomonitor the quality of river waters: the most commonly used options, aspects that require further research and recommendations proposed in this review paper are also shown. Methodological aspects 1. Selection and preparation of the bryophytes 1.1. Selection of monitoring species 1.2. Sample collection Selection of the reference site 1.3. Selection of material for transplant 1.4. Pre-exposure treatment 1.4.1. Washing with cellular extractants Number of washes Duration Shaking Weight moss / Volume of extractant 1.4.2. Washing with water Number of washes Duration Shaking Type of water Weight moss / Volume of water 1.4.3. Vital status 2. Preparation of the transplants 2.1. Type of transplant

Most commonly used option

Needs further research

Recommendations

Fontinalis antipyretica (55%)

Growth rate of the cultures

F. antipyretica or P. riparioides

Unpolluted site (39%) Entire shoots (71%)

– Comparison apical segments/ green parts

Laboratory culture Green parts of the shoot or apical segments (2–3cm)

–

Effect on accumulation capacity and variance 1 wash with EDTA (10 mM) 20 min Wash with shaking 1 L per 12 g dw

Wash with water (37%) 1 (16%) – – Deionized water (13%) – Live (97%)

–

Bags (74%)

Comparison between types of transplants

Once before washing with EDTA and twice after 20 min Wash with shaking Distilled water 1 L per 10 g dw Oven drying with a 50-80-100º C temperature ramp

2

2.1.1. Mesh size 2.1.2. Mesh net material 2.1.3. Size of transplant

6–30 mm (38%) Plastic (45%) 38-50 mg cm-2 (56%)

2.2. Pre-exposure storage 3. Exposure of the transplants 3.1. Location and support 3.2. Duration of exposure

< 24 h (18%) Attached to the riverbed (32%) 3–4 weeks (45%)

3.3. Number of bags per site

1 (21%) 1–3 (45%)

3.4. Initial concentrations and controls 4. Post-exposure treatments 4.1. Selection of material for analysis 4.2. Method of cleaning/ washing 4.3. Drying

Initial times (61%) and controls (4%)

4.4. Homogenization 4.5. Post-exposure storage

Weight of mosses /Surface area of the bag

16 mm2 Glass fibre, nylon or high density plastic 3.5 mg cm-2 At -20º C in individual sealed plastic bags

Effect of depth Influence of chemical, physical and biotic factors Local variation in the concentration of contaminants –

River bottom, midstream zone 2 weeks 3 at least 3 controls and 3 initial times

Apical segment 1–4cm (20%)

–

Selection of material before transplant

Wash with water (71%) 40ºC (26%)/ 2–3 days (21%)

Effect of washing with water –

Homogenization (11%) –

– –

No treatment Drying at 40ºC for 24 h plus drying an aliquot at 100 ºC to constant weight for accurate determination of dry weight of the material. Mill components made of titanium, zirconium or corundum Glass recipients

8. Conclusions

Nevertheless, the lack of standardization of the methods used hampers comparison of the results obtained in different studies and the use of the technique by environmental protection authorities as a tool for routine environmental monitoring. Although some aspects have not been thoroughly investigated, we propose a protocol that summarizes all of the recommendations discussed in this review paper. The different

Aquatic bryophytes are used as a simple, reliable and economic tool for active biomonitoring of water quality, thus enabling intensive surveys to be carried out and the monitoring of a large number of different contaminants, both organic and inorganic, in the same sample.

20

Number of publications (%)

Flat Bags

Fig. 5. Evaluation of the reviewed articles based on the degree of consistency between the methodologies applied in each and the recommendations proposed in this review. The scores vary between 0 (no information provided about the particular methodological aspect) and 20 (maximal consistency with the proposed recommendations).

10

0 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

Score awarded to methodology 468


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Debén, S., Aboal, J.R., Carballeira, A., Cesa, M., Real, C., Fernández, J.A., 2015. Inland water quality monitoring with native bryophytes. A methodological review. Ecol. Indic. 53, 115–124. Debén, S., Fernández, J.A., Carballeira, A., Aboal, J.R., 2016. Using devitalized moss for active biomonitoring of water pollution. Environ. Pollut. 210, 315–322. Diaz, S., Villares, R., Carballeira, A., 2012. Uptake kinetics of As, Sb, and Se in the aquatic moss Fontinalis antipyretica Hedw. Water Air Soil Pollut. 223, 3409–3423. Directive 2000/60/EC, 2000. European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. Off. J. L327 (22.12.2000). Engleman Jr., C.J., McDiffett, W.F., 1996. Accumulation of aluminium and iron by bryophytes in streams affected by acid-mine drainage. Environ. Pollut. 94, 67–74. Fernández, J.A., Vázquez, M.D., López, J., Carballeira, A., 2006. Modelling the extra and intracellular uptake and discharge of heavy metals in Fontinalis antipyretica transplanted along a heavy metal and pH contamination gradient. Environ. Pollut. 139, 21–31. Fernández, J.A., Aboal, J.R., Carballeira, A., 2010. Testing differences in methods of preparing moss samples: effect of washing on Pseudoscleropodium purum. Environ. Monit. Assess. 163, 669–684. Ferreira, D., Ciffroy, P., Tusseau-Vuillemin, M.H., Garnier, C., Garnier, J.M., 2009. Modelling exchange kinetics of copper at the water-aquatic moss (Fontinalis antipyretica) interface: influence of water cationic composition (Ca, Mg, Na and pH). Chemosphere 74, 1117–1124. Figueira, R., Ribeiro, T., 2005. Transplants of aquatic mosses as biomonitors of metals released by a mine effluent. Environ. Pollut. 136, 293–301. Gecheva, G., Yurukova, L., 2014. Water pollutant monitoring with aquatic bryophytes: a review. Environ. Chem. Lett. 12, 49–61. 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