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Science of the Total Environment 432 (2012) 143158

Contents lists available at SciVerse ScienceD irect

Science of the Total Environment

jou rna l hom e p a g e : w w w. e l se v i e r . c o m / l oca te /sci toten v

Review

Moss bag biomonitoring: A methodological review

A. Ares a,,J.R. Aboal a, A. Carballeira a, S. Giordano b, P. Adamo c, J.A. Fernndez a

aEcologa, Facultad de Biologa, Universidad de Santiago de Compostela, c/ Lope Gmez de Marzoa sn 15782Santiago de Compostela, Spain

bDipartimento di Biologia Strutturale e Funzionale, Universit di Napoli Federico II, Complesso Universitario MonteS. Angelo, Via Cintia 4, 80126Napoli, Italy

cDipartimento di Scienze del Suolo, della Pianta, dell'Ambiente e delle Produzioni Animali, Universit di NapoliFederico II, Via Universit 100, 80055 Portici (NA), Italy

a rt i c l e i n fo

Article history:

Received 1 February 2012

Received in revised form25 May 2012Accepted 25 May 2012

Available online 21 June2012

Keywords:

Moss bags

Active b iomonitoring

Airpollution

Technique standardization

a b s t r a c

t

Although the moss bag technique has been used for active biomonitoring forthepast 40 years, there is stillno standardized protocol that enables application of the technique as a tool to monitorair quality. The aimof this review paper is to evaluate the degree of standardization of each of the variables that must beconsidered in applying the technique (i.e. thevariablesassociated with preparation of the moss and mossbags, exposure of the bags, and post-exposure treatment). For this purpose, 112 scientificpapers thatreport the methods used in applying the moss bag technique were consulted. Finally, on the basis of theconclusions reached, wepropose a protocol that will enable each of these variables to be investigated

separately, with the final aim of developing a standardizedmethodology. 2012 ElsevierB.V. Allrightsreserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . 1442. Selection and preparation of the moss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 1452.1. Selection of species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1452.2. Selection of material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 147

2.3. Pre-exposure treatment and vital state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 147

2.3.1. Washing with water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1472.3.2. Devitalizing treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1472.3.3. Drying moss samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 148

3. Preparation of the transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 148

3.1. Mesh net material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1493.2. Mesh size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 149

3.3. Moss bag shape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 149

3.4. Quantity ofmoss used and size of bags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149

3.5. Storage of bags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 149

4. Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 149

4.1. Irrigation systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1504.2. Shading systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 150

4.3. Shelter systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. 1504.4. Positioning of mossbags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1504.5. Location and type ofsupport used . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://www.sciencedirect.com/science/journal/00489697http://www.sciencedirect.com/science/journal/00489697http://www.sciencedirect.com/science/journal/00489697http://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.elsevier.com/locate/scitotenvhttp://www.sciencedirect.com/science/journal/00489697http://www.elsevier.com/locate/scitotenvhttp://dx.doi.org/10.1016/j.scitotenv.2012.05.087


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. 150

4.6. Height of exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1514.7. Duration of exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1514.8. Numberof bags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 151

4.9. Initial and control concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 152

5. Post exposure treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 153

Corresponding author. Tel.: + 34 881816926; fax: + 34 981596904.

E-mail address: [email protected] (A. Ares).

0048-9697/$ see front matter 2012 ElsevierB.V. All rights

reserved. doi:10.1016/j.scitotenv.2012.05.087
mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.scitotenv.2012.05.087http://dx.doi.org/10.1016/j.scitotenv.2012.05.087mailto:[email protected]://dx.doi.org/10.1016/j.scitotenv.2012.05.087


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144 A. Ares et al. / Science of the Total Environment 432 (2012) 143158

6. Conclusions and final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 154

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

References . . ..

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

1. Introduction

Monitoring the air for contaminants is a complex technical task,and techniques for monitoring atmospheric contamination havebeen the objects ofintensive research in past decades (Helsen, 2005).InEuropean countries, air quality measurement is based onphysico-chemical techniques. These techniques can provide accuratemeasurements of some principal atmospheric pollutants (mainly CO,SOx, NxOy, PAHs and particulate matter) within relatively short

periods(i.e. hours ordays). How- ever, there are technical difficultiesin measuring other contaminants included in the EuropeanParliament Directive (2008/107/EC) relating to air ambient quality(i.e. As, Cd, Hg, Ni, Pb),and analysis ofthese contaminants in air isvery expensive. These contaminants are potentially highly toxic, asdemonstrated by toxicological and epidemiological studies (see e.g.Kongtip et al., 2006;Yang and Omaye, 2009). An alternative way of

monitoring these contaminants is to use terrestrial mosses (or otherorganisms) as biomonitors. The use ofmossesenables simulta- neousmonitoring of a large number of contaminants (i.e. metals andmetalloids, PAHs, radionuclides) with the same sample, and also hasotheradvantages over current methods (e.g. simplicity,reliability, costeffectiveness and the lack of the need for electricity). Furthermore,biomonitoring with moss is inexpensive, and can provide a goodprelimi- nary overview of the state of the air quality because densesampling networks can be used. Nevertheless, there are somedisadvantages associated with biomonitoring, such as the factthat information is only obtained afterrelatively long periods. Otherdisadvantages are discussed throughout the different sections of thisreview paper.

Among the other terrestrial organisms employed so far asbio- monitors of air pollution, lichens have been often used incomparison with mosses. Despite lichensproved a better resistanceto environmental stress, preserving or recovering their vitalityduring biomonitoring, most studies have highlighted a lichenlower capacity to intercept and accumulate most of the airborneelements compared to mosses (Spagnuolo et al., 2011). Differencesin surface structure have beensuggested as one of the main factorsexplaining thisdiver- sity (Adamo et al., 2007). This review doesnot intend to compare moss with lichen biomonitoring, whichalone deserves a specific ef- fort. Nevertheless, along the text someconsiderations on the peculiar performance ability of the lichens areemphasized.

Two types of biomonitoring are clearly differentiated in theliterature on the use of mosses to evaluate atmosphericcontamination: (i) passive biomonitoring, using moss that grow

naturally in a particular area, and (ii) active biomonitoring, bytransplanting moss from other locations. Whilst the use of nativemoss is more appropriate for extensive studies in large areas (i.e.regional or national studies), active biomonitoring is more usefulfor intensive studies in smaller areas (i.e. urban or industrialareas). For activebiomonitoring, moss samples are collected fromrelatively unpolluted habitats; they are then cleaned, selected and

pre-treated before being exposed in a different environment. Theuse of moss transplants resolves various problems associated withthe use ofnative moss. Firstly, it overcomes the scarcity orabsence ofmoss in certain environments (i.e. industrial and urban areas).Secondly, it reduces the high degree of variability in the uptake ofcontaminantsby native moss within the same sampling site (Aboal etal., 2006; Fernndez et al., 2002). According to Castello (1996)and

Varela et al. (2010), the coefficients ofvariation(CV) for aparticularelement are lower between replicates of moss transplants than

between subsamples of native moss at a sampling site (e.g. the CVfor Pb in moss bags ranged between 14 and 21% for 10 replicates

exposed for 2 months, in comparison withaCV that ranged between


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33 and 93% for 50 samples ofautochthonous mosscollected at a sam-pling site). Thirdly, transplants canbe used more conveniently for inter-pretation of the temporal variability in the results. When moss bagsare used, the initial concentration and exposure period are known.However, the concentrations in native moss areassumed to representthe contamination corresponding to a particularsite, without takingthe temporal variability into account, and highly variable results havebeen obtained in short periods of time, i.e. 34 days (Boquete et al.,2011a). Fourthly, transplants minimize the possible edaphic influenceon the concentrations of certain elements (see e.g. Bargagli etal., 1995; Berg and Steinnes, 1997). Transplants are usually

exposed at a certain height above the ground,but naturally growingmoss is subjected to rain splash and accumulation of soluble soilcompounds during periods when there isclosecontact between soil andwater (Berg and Steinnes, 1997). Final- ly, any phenotypic and/orgenotypic adaptation that may take place in contaminatedenvironments will modify the tissue concentrations ofcontaminants(Fernndezet al., 2000; FernndezandCarballeira, 2000; Tabors et al.,2004); this problem can be solved by transplanting moss fromuncontaminated areas.

The effect of contaminants on the physiology ofnative mosses isdifficult to evaluate, and it is impossible to isolate theireffects fromthose ofotherenvironmental variables. However, the use of irrigatedmoss transplants removes certain environmental stressors (e.g. hy-dric stress or stress from solarradiation), and thus isolates the effect

ofcontamination.The moss bag technique is the most common typeofactive bio-monitoring with terrestrial mosses that is reported in theliterature. The technique, which was originally introduced byGoodman and Roberts (1971), involves exposure of moss samplesheld within mesh bags in order to monitor the presence ofcontaminants in the air. In the literature consulted (112 papers

published between 1971 and 2011, lo- cated through SciVerseSCOPUS), the technique was mainly used to monitor levels ofinorganic contaminants. Thus, most studies (86%) monitored levels ofmetals and metalloids, and far fewer monitored levels of organiccontaminants such as polycyclic aromatic hydrocarbons (PAH) (4% ofthe studies), polychlorinated biphenyls (PCB) (1%) and others (9%).Of the metals and metalloids studied, Pb was determined in 67studies, followedby Zn (55), Cu (52), Cd (49), Fe (46) and Ni (43).

However, although the moss bag technique has been used for ap-proximately 40 years, standardized protocols have unfortunatelystill not been developed. Standardization of the following stepsshould be considered: (i) preparation of the moss; (ii) preparation ofthe transplants; (iii) exposure of the transplants; and (iv) postexposure treatment. The low degree of standardization reached inthe technique is reflected by the fact that some studies provide verylittle or contradictory information (e.g. Kupiainen and Tervahattu,2004) or sometimes even inaccessible information (Lodenius, 1998)about the methods used. Some authors even claim to have usedstandardized transplants, although they have not followed therecommendations given in previously published papers(see e.g. Solgaet al., 2006; Temple et al., 1981). The lack of such protocols hinderscomparison ofthe results obtained in different studies, and sometimes

limits the conclusionsthat can be reached.The first authors to apply the technique (Goodmanand Roberts,

1971) used procedures that were not based on the results ofprevious research. The first modifications were introduced onlythree years later (Little and Martin, 1974), and were followed byseveral othermodifications, so that several ways of applying thetechnique have been reported. These range fromsimplemethods inwhich the moss tissues are barely handled (e.g. transplantation ofmoss mats, see e.g.Acar, 2006; Samecka-Cymerman and Kempers,2007) tomethods


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Numberofpapers

A. Ares et al. / Science of the TotalEnvironment 432 (2012) 143158145

involving complex pretreatment and exposure systems (see e.g.Amblard-Gross et al., 2002; Couto et al., 2004a; Rivera et al., 2011).As a result, many methods have been used only once by thesame group of researchers. Furthermore, as some 68% of theauthorshave only published one paper on the topic, numerous methodshave been reported (Fig. 1). Therefore, despite the advantagesassociated with the technique, its use has been limited toscientific research, and it has not been implemented by public

authorities to monitorat- mospheric contamination. Moreover, useof the technique is largely limited to Europe (i.e. 80% ofpublished

papers; Fig. 2).Despite all of the above, several authors have focused their

research on methodological aspects,with the aim ofestablishing astan- dardized methodology. The first studies of this type werecarried out by Gailey and Lloyd (1986b,c,d), who investigated therelation between weight and surface area of the moss, and theoptimal time ofexposure in moss bags (Fig. 3). Unfortunately,their conclusions were scarcely noted in subsequent studies. Inthe following years, very few studies addressed methodologicalquestions, but from2007 onwards (i.e. in 60% of the studies relating to themethodology;Fig. 3), a small numberofresearcherswhoaddressed this problem fo-cused their research on aspects such as the treatment of themoss prior toexposure (Adamo et al., 2007, 2008a; Giordano et al.,2009; Fernndez et al., 2010; Tretiach et al., 2007).

The present review considers four key aspects inrelation to stan-dardization of the technique of active biomonitoring of airquality: (i) selection and preparation of the moss tissue; (ii)preparation ofthetransplants; (iii) exposure of the transplants; and(iv) post exposure treatment of the transplants.Afterreviewing anddiscussing the literature consulted, we discuss whether the

particular stage of the process can be standardized or furtherresearch is required.

The overall aim of the present paper was to review a seriesofarticles dealing with the moss bagtechnique, in orderto proposea standardized protocol for preparing and exposing mosstransplants that meets the following requisites: (i) that the

transplants are easy to prepare and handle; (ii) that they enablereplicable results to be obtained; (iii) that they are capable ofcapturing high concentrations of as many contaminants as possible;and (iv) that they are efficient at capturing contaminants, and arecapable of indicating their occur- rence in the air within areasonable period oftime.

2. Selection and preparation of themoss

Preparation of the moss prior to transplantationalways includesselection of the species and usually selection of the portion of theshoot, as well asdiversepre-exposure treatments.

12

10

8

6

4

2

00 50 100 250

Number of authors

Fig. 1. Numberofpapers on active biomonitoring ofair quality with terrestrial moss,

in relationship tonumberof authors.


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2.1. Selection ofspecies

The particular species used will greatly affect theresults(Castello,2007; Culicov and Yurukova, 2006), and comparable results may notbe obtained with different species. However, although many differentspecies (i.e. 45) have been used to date, in some cases thisinformation is omitted (Tan et al., 2000) or only the genus isindicated (see e.g.: Carpi et al., 1994; Santamara and Martn, 1997;Wegener et al., 1992). Thus, approximately 55% of the species haveonly been used on one occasion. Given this diversity, we havegrouped the genera to show the frequency with which they have

been used in previous studies (Fig. 2). Moss bags have most oftenbeenprepared with species of the genus Sphagnum, followedby othergenera widely used in Europe, represented by Hypnum cupressiformeHedw., Pseudoscleropodium purum (Hedw.) Fleisch and less oftenPleurozium schreberi (Brid.) Mitt. and Hylocomium splendens (Hedw.)Schimp. The main reason for selecting a particularspecies appears tobe its presence and abundance in the study region, which hasenabled the simultaneous use of native and transplanted moss insome studies (see e.g.Fernndez and Carballeira,2000; Kosioret al., 2008; Ratcliffe,1975).

Selection of the species should take the followingcriteria into con-sideration: (i) preference for widely distributed and pleurocarpousmosses; (ii) the selected species should have some structural and

physicochemical characteristics that will allow efficient uptake of

contaminants from the atmosphere; and (iii) the selectedspecies should be one of the most commonly used and about whichmost information exists.

The availability of large specimens will facilitatehandling (i.e. se-lection of the portion of shoot to beexposed, cleaning, etc.) and re-duce the possibility ofloss of material during exposure. In addition,thecapacity of the different species of moss to capturecontaminantsdepends on the following: (i) the morphology of the shoots, whichdetermines the capacity of the moss to retain particles, as wellas the circulation of gases and water around the tissues; (ii) thecation exchange capacity (CEC), which is mainly linked to thequantity ofuronic acids in thecell wall and the outer parts of the cellmembrane, andtheir degree ofmethylation (Carballeira et al., 2008;

Richter and Dainty, 1989); and (iii) the specific surface area (i.e. m2

kg

1

), as ob- viously the area exposed to the environment willincrease with the surface area and roughness of the tissues. Thecapacity of the moss to accumulate elements will thereforeincrease with the numberofphyllids per caulid (Sun et al., 2009).Clough (1975) concluded that the rough surface and hairiness ofmoss reduce subsequent removal of elements by blow-off, bounce-off and rain wash. Adamo et al. (2007) explained the higherefficiency of metal accumulation in H. cupressiforme than in thelichen Pseudevernia furfuracea (L.) Zopf on the basis of the larger

specific surface area of the moss (rangingbetween 81 and 136 m2

kg1

).

Very few ofthe studies reviewed have compared themorphologicaland physicochemical characteristics of different transplanted species,and the only information available in this respect refers to thefinal effect of these characteristics on the bioconcentration ofelements after transplantation of the moss samples in the samelocations within the same exposure periods. In regard to studiesthat compare the most commonly used types of moss, Ratcliffe(1975) concluded that the concentration of Pb in bags of H.cupressiforme was only 66% ofthat in bags of Sphagnum spp. Thiswas confirmed by Culicov and Yurukova (2006) on comparing H.cupressiforme and Sphagnum girgensohnii Russow. Furthermore,Castello (2007) exposed spherical bags of H. cupressiforme and P.

purum simultaneously in an industrial environment, and concludedthat the latter was a better accumulator, showing similar or higheraccumulation and lower loss of almost all elements than the former.Otherauthors such as eburnis and Valiulis (1999) demonstrated thatthere was no significant difference between H. splendens and P.schreberi as regards quantitative uptake of metals. Tremper et al.

(2004) compared P. schreberi and Rhytidiadelphus


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51

Numberofpa

pers

1975

1980

1985

1990

1995

2000

2005

2010


124

943

1 99

2

132

6 14 11

1

2

Genera ofmossused Genera employed in theworld Frequency ofuse

Sphagnum sp.

Hypnum sp. Rhytidiadelphus sp. 29

Pseudoscleropodium sp.

Pleurozium sp.

Thuidium sp. 12

Calyrnferes sp.4

9Others


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However, culture of Physcomitrella patens (Hedw.) Bruch & Schimp.(by vegetative propagation of the filamentous juvenile protonematissue in suspension culture) has been optimized, therebyenabling large scale production of biomass of the species for

biotechnological purposes (Decker and Reski, 2004, 2007, 2008).However, both C. purpureus and P. patens are small acrocarpousspecies and their morphology and size may determine structuralcharacteristics (i.e. surface area to mass ratio) that would make

them unsuitable forhan- dling andcapture ofcontaminants.With the available information regarding the moss species most

commonly used in active biomonitoring,any ofthe most common bio-monitoring genus (Fig.2) are suitable forthe use as a standard but spe-cies of the genus Sphagnum best fit the criteria for the suitability ofmoss species in the moss bags technique. They are of a suitable sizefor handling and have been cloned (Sstad et al., 1998), althoughthey have not been cultivated for use in moss bags. The

physicochemicalproper- ties of Sphagnum also favour their use: theleaves constitute about two thirds of the dry biomass andprovide avery large surface area forcation exchangeprocesses and capture offine airborne particles (Bargagli,1998); the hyalocysts have large pores that can trap airborneparticulate (Giordano et al., 2005); and the cation exchange capacity

has been esti- mated to be 0.91.5 meqg 1 dry weight (i.e. higher

than that ofothermosses: 0.61.1 meq g1)(Temple et al., 1981).

2.2. Selection ofmaterial

Once aparticular species has been selected for use inthe moss bagtechnique, the next step is to decide which portion of the shootsshould be used. The results may vary depending on the materialselected, because the older parts of the stems will containdifferent amounts of some elements than younger tissues (Tavaresand Vasconcelos, 1996; Leblond et al., 2004; Fernndezet al.,2010).However, this information was not provided in 56% of all the papersreviewed, and in the otherpa- pers (44%) the following options werechosen: apicalportions of the shoots (19.4%), green parts (10.2%)andentire shoots (14.3%).

The aim of selecting the material is to obtain transplant materialthat is as homogeneous as possible. The shoots of naturally growingmoss are highly variable in size, and therefore the portion ofolderand green parts will also vary. As the concentrations in these

portions will differ (see above), and their capacity forbioconcentration will probably also differ, the use of whole shootsshouldbe discounted.

Despite the different substrates and exposure toatmospheric de-position, green shoots of epiphytic and epilithic H. cupressiformefrom the same site show a rather homogeneous chemical composi-tion (CV b 20%) as regards most majorand trace elements,with themain exceptions being concentrations of Cr and Ni (CV 43 to57%) (Adamo et al., 2008a). The concentrations of Cd, Mg and Znwere higherin theepilithic moss, which is more exposed to soil and

rockdust, than in moss that grow on tree trunks.Different authors argue in favour ofthe use ofapicalportions of sim-

ilar size (Fernndez and Carballeira, 2000; Gailey and Lloyd, 1986b;Gill et al., 1975; Little andMartin,1974) because the exclusive use ofsuch portions eliminates one source of variability and ensurescomparable exposure times. However, no authors have argued infavour of the use of whole shoots. There are two disadvantagesassociated with the use of whole shoots. Firstly, their variable sizeimplies that the capacity for accumulation will also vary due to theheterogeneousnature of the material; secondly, identification of thegreen part is somewhat subjective.

The availability of green shoots of similar size and with similar con-centrations of elements and capacity for bioconcentration isanother potential advantage associated with the use of laboratory-cultured moss.

2.3. Pre-exposure treatment and vital state

Once the material has been selected, it can either be treated ornot treated prior to exposure. As the vital state of the moss (live ordead)


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will depend on the prior treatment, both aspects will beconsidered together. The aim of such treatment is to obtaintransplants with similar and well defined initial features (similarmorphology, well- characterized initial contents of contaminants,comparable physiological status; Giordano et al., 2009), and tomaximize thebioconcentration capacity (Gailey and Lloyd,1986b). In81% of the papers reviewed, a treatment prior to exposure wascarried out. Some of the treatments used (acid washing and/or ovendrying) lead to death of the moss. When the moss is subjected tosome type ofpre-exposure treatment, in 78% of the cases the mosssamples were exposed in a live state, and in 32% the moss samples

were killed. The sum of these percentages is greater than 100%because both options weresometimes used in the same study.

2.3.1. Washing withwaterIndependent of the vital state of the moss whenexposed, any pre-

exposure treatment generally included washing with water (72% ofthe papers). The objectives ofwashing are: (i) to clean the moss ofedaphic particles and/or plant remains, so that the initial concentra-tion of elements is as homogeneous as possible (Tretiach et al.,2007; Adamo et al., 2008a), and (ii) to partially activate the tissues,by removing some elements bound to cation exchange sites on thecell wall and membrane(Fernndez et al., 2010).

The outcome of the washing step is determined by diverseparam- eters: (i) number of washes; (ii) duration of washing; (iii)

use (or not) of shaking; (iv) type of water; and (v) relationbetween the weight of the moss and the volume of water.However, information about all of these parameters was onlyreported in one of the papers reviewed (Tretiach et al., 2007),whereas in 42% of the papers, none of the above parameters wasspecified. The number of washes, the most specific aspect (53%),ranged between 1 and 7, with the most common being 3 (47%)and 7 (22%). The duration of washing was only reported in 10% ofthe studies, and was extremely variable (ranging between oneminute and overnight), and also varied when more than onewashing step was included. Shaking during washing was onlyapplied in 12% of the studies, and details of the intensity were not

provided. The type of water was not usually specified (25%), butwhen specified it was usuallydistilled (80%) orbidistilled (13%). Inone study,methanol was used in addition to distilled andbidistilledwater to wash the samples (Strachan andGlooschenko, 1988). Final-ly, in the few studies inwhich the weight of the moss portions in re-lation to the volume ofwaterwas specified, it was 100 g dw per10 Lof water (Adamo et al., 2007, 2008a,b; Giordano et al., 2009,2010; Tretiach et al., 2007, 2011). Taking into account all of theabove, there is not sufficient evidence to enable selection of theoptimal values ofthe parameters considered.

In any case, even when the moss is washed with waterbefore expo-sure and is expected to perform as a livingorganism, the samples some-times die during exposure, as a consequence of dry and stressfulenvironmental conditions (Adamo et al., 2003; Giordano et al., 2005;Tretiach et al., 2007). Accumulation of gaseous S, N and Ccontaminants is greatly influenced by the vitality of the biomonitor(Vingianiet al.,

2004). Inpapers in which live vs. dead mosses andlichens were com-pared as active biomonitors, livingmaterials did not generally showa better performance than dead materials (Adamo et al., 2007).Water washed lichens and mosses had statistically lower orstatisticallynot significant increments in their trace element content afterexposure in two Italian cities, Trieste and Naples. An interesting

pattern was detected with respect to C and N, because only waterwashed lichens (whose vitality was not excessively affected byexposure, see Tretiach et al.,2007), significantly accumulate C and N during theexposureperiod.

2.3.2. DevitalizingtreatmentsThe use of live or dead moss depends on theobjective of the par-

ticular study, which may be tobiomonitor atmospheric contamina-

tion or to study the effects of such contamination on moss. For the


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first objective, devitalization enables the efficiency of contaminantcapture to remain constant, as capture ismainly due to passive uptake

processes that are independent of the vitality of the moss (Aniiet al., 2009a, 2009b; Basile et al., 2009; Giordano et al., 2009). Thesepro- cesses also include the cation exchange capacity and thecapacity forparticle retention. The latteris of great interest in urbanand industrial areas where particulate forms predominate inemissions (Castello,2007), and the technique is frequently used in such areas. On theother hand, the use of dead samples has some advantages over theuse of live moss: (i) from a practical point of view, moss bagscan be pre-prepared and will therefore be readily available at alltimes; (ii) the results will be less variable (Adamo et al., 2007;Castello,1996; Gailey and Lloyd, 1986d; Giordano et al., 2009); and (iii) mossmetabolism will not affect the results (Giordano et al., 2009). In thelatter respect, Fernndez et al. (2010) have recently demonstratedthat growth of P. purum during the exposure period affects metaluptake and generates differences in the concentrationsdepending onthe mea- sured element. Some elements (i.e. Cd, Cu and Zn) showhigher concentration in theportion grown during the exposureperiod whereas other elements (i.e. Hg and V) show higherconcentration in the original portions, independently of

contaminant uptake during the exposure period.Although some attention had already been given to devitalizing

treatments in the past (Castello, 1996; Gailey and Lloyd, 1986b;Tavares and Vasconcelos, 1996), this has increased in recent years(Adamo et al., 2007, 2008a, 2011; Giordano et al., 2009; Tretiach etal., 2007).

2.3.2.1. Acid washing. Acid washing (also called activation) is thedevitalizing treatment mostcommonly used (25% of all studies and78% of the studies in which the moss samples aredead). This treat-ment consists of immersion of theselected moss material in an acidmedium, with the aim of leaching metal ions from the cellwalls (Adamo et al., 2007; Castello, 1996; Gailey and Lloyd,1986d)and dis- rupting biological membranes (Brown and Brown, 1991;

Brown and Wells, 1988). This regenerates the cation exchange sitespresent on the cell wall (i.e. converts them into hydrogen (H+)form),with the aim of increasing the bioconcentration capacityof the exposed moss. Adamo et al. (2007) showed that for H.cupressiforme, acid washing can reduce the Pb present by up to96%, relative to oven dried samples, and by 94% relative tosamples washed with water. As noted for washing with water, theresults of the acid treatment aredeterminedby various parameters:(i) number ofwashes; (ii) duration of washing; (iii) use (or not)of shaking; (iv) acid used and concentration; and (v) ratio

between the weight of the moss and the volume of the acidsolution. In 26% of the studies in which the samples are washed inacid solutions, none of the above parameters was specified. Themost common method is a single washing step, and in only 11%of the studies, the samples were washed 3 times. The duration ofthe washing step was only specified in 19% of the studies, andwas extremely variable ranging between 1 and 96 h in total(including all washing steps). No studies specified whether thesamples were shaken during treatment. The acid most commonlyused is HNO3;theconcentrations used range between 0.025 and 1 M,

with 0.5 M being the most commonly used (in 54% of thestudies). The next most commonly used acid is HCl; concentrations of

between

0.01 and 0.5 M are used, with the latterconcentrationthe most com-monly used in the studies in which this was specified (80%). With theexception of the study byAdamo et al. (2007), in which 1 mL of 1 MHNO3 solution was used for 2 mg of moss material, no otherstudies

specified the ratio between the weight of the moss and the volumeof the acid solution.

2.3.2.2. Oven drying. The use of oven drying as a method ofdevitalizing the moss is much less common (7% ofallstudies and 23%

of studies in which moss is dead); the method consists ofmaintaining the material


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in an oven at a high temperature (> 100 C) for 24 h. Althoughthe aim of this treatment is to devitalize the moss, the moss mayactually be activatedby thistreatment as some of the metals may bevolatized at high temperatures. In 86% of cases, the methodrecommended by Giordano et al. (2009), i.e. drying the moss at120 C for 24 h, was followed. In some studies in whichtemperatures between 40 and50 C were used (Dmuchowski and Bytnerowicz, 2009; Rivera et al.,2011), it was not clear if the aim of the drying process was todevitalize the moss. The advantages ofoven-dryingoveracid washinghave been described by Giordano et al. (2009): (i) it is eco-friendly,

and (ii) it leaves theparticular morphology of the moss and leafletarrangement virtually unaltered, without changing the capacity forcapture. Washing with acid notably deteriorates the moss, leading torupture of the tissues. However, the disadvantage of oven-drying isthat the shoots of some mosses, such as Sphagnum, becomebrittle andthe small leaf frag- ments tend to fall off the stem. In our experience,drying mosses inside the mesh bags by gradually increasing thetemperature (to 120 C) minimizes the loss of material. Anotherdisadvantage is that drying does not release metals bound to cationexchange sites. This may be resolved by the use of chelating agentssuch as EDTA (Lodenius and Tulisalo, 1984), penicillamine and/ordimercaprol (Prez-Llamazares et al., 2010) prior to washing withwater(see Section 2.3.1).

Comparison of the different pre-exposure treatments (washing

with water, acid washing and oven-drying) applied to H.cupressiforme did not reveal any trends in terms of the efficiencyof accumulation of chemical elements, i.e. it was not possible toidentify the optimal treatment. This has been attributed to thefact that the surface texture remains basically unchanged afteroven-drying and acid washing, as surface interception of air-borne

particulate is likely to play a major role in accumulation in mosstransplants in the urban environment (Adamoet al., 2007; Tretiachet al., 2007).

2.3.3. Drying mosssamplesIn many of the studies (41%), the final step inpreparing the mate-

rial prior to producing the moss bags consists of drying at low tem-perature (ranging from room temperature to 40 C), which does notcause death of the moss. The aim of this dryingprocedure is to re-move excess moisture that may remain on the moss afterwashing with water or acid (see Sections 2.3.1 and 2.3.2.1). Inalmost all of thepapers reviewed, no information was given abouteitherthe temperature or the duration of the drying step.

3. Preparation of thetransplants

Preparation of the moss transplants usually involvesplacing themoss in some type of support (e.g. a mesh net bag), althoughin some cases moss mats are transplanted (7% of studies). In thelatter case, preparation of the transplants, handling of whichshould be minimal, consists of extracting a portion of the mossfrom a certain area (usually unpollutedzones) and transplanting it inanother area (usually contaminated zones). The transplanted moss

can beplaced within a frame, e.g. made of wood (Naszradi et al.,2004) or polyethylene (Boquete et al., 2011b), or can be placedamong the native vegetation without any type of frame (e.g.Huttunen et al., 1981). Such transplants are often used to evaluatethe degree ofadaptation of native moss to the contamination in a

particulararea (see e.g. Kosior et al., 2008, 2010; Tabors et al., 2004).Therefore, the aim of such studies is different from that of moss bagstudies, and the methodology is more similar to that used withnative moss monitoring.

When preparation of the transplants involves placing the moss ina mesh net bag, the results obtained will beaffectedby the character-istics of the bag. Such characteristics include the composition andsize of the mesh net, the form and size of the bag and the useof auto irrigation systems. The different alternatives for each of

these characteristics, as reported in the literature, are reviewed inthe following sections.


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3.1. Mesh net material

The composition of the mesh net material is usuallyspecified (91%), and is usually a type ofplastic such as nylon (71%),

polypropyl- ene (5%), polyethylene (3%) or non-specified plastic(17%). The materials areusually produced commercially for diverseuses such as hair nets or mosquito nets. Selection of materials suchas plastic or glass fibre is more suitable than other less frequently

used materials such as cotton (3%) or metal (1%), which mayminimally interfere with theuptake process.

The mesh nets are sometimes washed with diluteacidbefore pre-paring the transplants, to remove trace contaminants (Fernndez etal., 2000). This particularlyapplies to mosquito nets, which are some-times impregnated with insect repellents. Finally, the bags areusually closed with nylon fibre.

3.2. Mesh size

Of the papers reviewed, 45% did not specify themesh size. In theremaining papers, the distribution of the mesh size (in size classes,

mm2) is extremely variable: b 1 (4%), 12 (36%), 24 (14%), 45

(28%), 410 (6%), 10100 (4%), 100150 (8%), and 150250 (2%). Se-lection of the mesh size is a compromise between maximization ofthe interception of aerial deposition and minimization of the risk ofloss of material. Inadequate selection of the mesh size may leadto loss of large amounts of moss as a result of wind action on themesh bags. For Sphagnum moss, such losses were quantified byStrachan and Glooschenko (1988) as between 50 and 75% of theinitial amount. The thickness of the mesh may also affect thecapture of contaminants by the moss because the mesh mayintercept and retain particulate material (Adamo et al., 2008b;Archibold, 1985; Cameron and Nickless, 1977; Zechmeister et al.,2006a). In a recent work, lichen samples exposed without nylon

bag (naked lichen), showed a lower accumulation of mostanalyzed elements than lichens exposed in bags (water washedlichens), although the difference was not statistically significant.

Nevertheless, the coefficient of variation of element concentrationwas higher in naked lichen than in water washed lichen,suggesting the net has a homogenising effect on elementaccumulation, probably acting as a sieve for airborne particulates,and enhancing thebouncing off of the largest fraction (Giordanoet al.,2012).

3.3. Moss bag shape

Although a wide variety ofshapes of moss bags areused, these canbe grouped in three categories:spherical moss bags (36% ofstudies),flat, square orrectangularmoss bags (23%), and cylindrical moss bags(3%). The shape of the bag was not specified in 39% of the

papers reviewed. The advantage of three-dimensional bags (i.e.spherical and cylindrical) is that they allow uniform collectionefficiency from all directions, whilst permitting collection bygravitational sedimentation (Little and Martin, 1974). In twodimensional bags (i.e. flat moss bags), exposure ofthe moss to theatmosphere is more uniform, and capture of elements from theatmosphere is improved by the use of some type of system to

prevent displacement of the moss further inside the bag (e.g. bysewing the bag to make compartments). This prevents the mossfrom being flattened on the bottom of the bag under certainconditions (e.g. rain, wind, etc.). The only study in which thesetypes of bags are compared was that carried out by Gailey andLloyd (1986d), who concluded that the moss in spherical bagscaptured higherconcentrations ofmetals.

The shape ofthe bags in auto irrigated transplants (seeSection 4.1)is determined by the type ofauto irrigationsystem used. This systemis sometimes included withinthebag (e.g. Couto et al., 2004a).

3.4. Quantity of moss used and size ofbags

In planning experiments with moss bags, thefollowing should beconsidered: i) verification of the availability in natural environmentofsufficient mossmaterial with reference to the experimental design(i.e.numberofpoint measures planned); ii) ensuringenough materi-al because of analytical constraints and (also because of) iii) loss ofmaterial during the exposure. Temple et al. (1981) reported an

averageweight loss of 15% in moss (Sphagnum sp.) due tolaboratoryhandling and deterioration ofsamples in thefield.

Gailey and Lloyd (1986b) reported that the optimal amount ofmoss that shouldbe included in each bag is 100200 mg. However,larger amounts are preferred to ensure sufficient material for chemi-cal analysis(andwhere repetition of the analysis is necessary) and toovercome the loss ofmaterial during handling andexposure. Quanti-ties ofbetween 400 and 500 mg perbag are often used (e.g. Adamoet al., 2003; Tretiach et al., 2007; Giordano et al., 2009). Accordingto Adamo et al. (2007), the efficiency of metal accumulation bydifferent biomaterials could be better compared by expressing theelement content of the biomonitor on a surface area basis ratherthan on a weight basis. Therefore, comparable amounts ofdifferent moss species in terms of specific surface area, expressed

in m2

kg

_1

, may be useful for defining the amounts thatshould beused.Once the amount of moss material has beenestablished, the size

and shape of the bag must beselected. The size of the bag determinesthe ratiobetween the weight of the moss and the surface area ofthebag. This ratio will affect the efficiency with which the transplantscapture contaminants from the atmosphere. However, this informa-tion is only provided in one of the 112 studies reviewed(Lodenius and Tulisalo, 1984), and in most studies (58%) the ratiocould not be calculated from the information given. In those studies

in which the ratio could be calculated (mg cm2), it was fairlysimilar: b 40 (76%), 4070 (9%), 70110 (9%) and >110(5%).

For most contaminants, the optimal size of the bagmust enhancemoss interception and uptake capability as well as replicability of theresults. Different authors (Gailey and Lloyd, 1986a; Zechmeister etal.,

2006b) recommend that the moss should be looselypacked in thinlayers as this minimizes overlapping and compression of the mossshoots, and enables even exposure of the material to thecontaminants. In fact, when the shoots overlap, theconcentrations will decrease gradually. In this respect, Temple etal. (1981) exposed flat bags with moss weight/bag surface arearatios of 10, 20, 30 40 and50 mg cm 2, and found that the bags with a weight/surface area

ratio of 30 mg cm 2 were the most suitable for the maximumuptake.

3.5. Storage ofbags

Once the transplants are prepared, they are storedbeforebeing ex-posed in the study area. Although information about storage of mossbags is scant, someauthors recommend storing the moss bags in indi-vidual sealed plasticbags (with e.g. a zip-locksystem) toprevent lossofmaterial and contamination ofthesamples.Regarding to the storagetemperature of thesamples, low temperatures (i.e. 20 C) seems to

be the adequate to prevent any degradation by bacterialactivity (Morita et al., 1997).

4.Exposure

The prepared transplants are exposed using different systems of

irrigation, shading and cover, type ofsupport,location, height and du-ration of the exposure and numberof bags exposed. The combinationof all these options will affect the final concentrations of contami-nants in the moss. As several differentcombinations are reported in


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the literature, the options will be considered separately.


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4.1. Irrigationsystems

Moss samples are usually exposed live (in 65% of thestudies reviewed). An irrigation system was used to keep the mossalive during the period of exposure in only 23% of such studies.Although some authors use non irrigated transplants (e.g.Cameron andNickless,1977; Hynninen, 1986; Lodenius and Tulisalo, 1984), manyothers consider that this method is not valid.The main problem isthat non irrigated transplants tend to dry out and thus theirefficiency in retaining metals will vary depending onenvironmental conditions (Giordano et al., 2009), such as airhumidity, precipitation, solar radi- ation, wind intensity, etc. (Tyler,1990). In some studies, higherconcentrations of metals have beendetected in wetseasons than in dry seasons (Adamo et al., 2003;Giordano et al., 2009; Tavares and Vasconcelos, 1996). However,the velocity of deposition may be higher in dry moss bags thanin wet moss bags because the more open structure of dry moss

bags provides a larger effective surface area for deposition(Clough, 1975), although for particles of > 5 m diameter,deposition velocities are higher in wet moss bags due to re- duced

bounce-off of particles from the wet surface. Some authors havereported death of the moss during the exposure period (Basile et

al., 2009; Goodman and Roberts, 1971; Huttunen et al., 1981;Tavares and Vasconcelos, 1996; Tretiach et al., 2007). This wouldlead to a change in the capacity of the moss to capture contaminantsat some timeduring the exposure period, thus preventing compari-son of the results obtained under differentenvironmental conditions(i.e. in time and space).Vingiani et al. (2004) demonstrated that, un-like trace elements, which are mainly accumulated by mosstransplants through passive processes of retention, theaccumulation of S, N and C by mosses is mainlybased on activeintake of gaseous forms of contaminants (i.e. SOx, NOx, CO andVOCs).

The alternative to using live, non-irrigated transplants is to use ir-rigated transplants. As already indicated,metal uptake is affected bythe moss metabolism, andexposure of the irrigated moss is only rec-

ommended when the aim is to study biological variables (e.g.enzyme activity, photosynthesis, etc.). Two types of irrigationsystems can be applied. The first, auto irrigation, was introducedbyAl-Radady et al. (1993); this system involves placing the moss on acapillarymat con- nected to a container, usually made ofplastic, fullof water. In some studies the moss is placed directly on acapillary mat and both areplaced in a mesh bag (Amblard-Grosset al., 2002; Couto et al.,2004a), whilst in other studies the meshbag containing the moss is

placed on the capillary mat (e.g. Al-Radady et al., 1993; Anii et al.,2009b). Higher concentrations have been detected in auto irrigatedSphagnum girgensohnii samples than in dry samples (Anii etal.,

2009b), possibly because the speed of deposition of particles ismuch higher on wet moss bags than on dry ones (Clough,

1975). The second type ofirrigationsystem, possibly less practicallyapplica- ble, is to spray the moss bags with distilled water once aweek(Basile et al., 2008) or twice a week (Mariet et al., 2011).

4.2. Shadingsystems

In six of the studies in which auto irrigated transplants were used,the samples were placed in a steel frame andcovered with a shadingnet,to reduce environmental and hydric stressors, such as direct solarradiation (reducedby up to 70%) and wind (Aboal et al., 2008;Couto et al., 2004a,b; Fernndez and Carballeira, 2000; Fernndezet al.,2010; Skert et al., 2009). The aim ofremovingenvironmental stressors

is to enable clearer identification ofrelationships between the concen-trations ofcertaincontaminants in moss tissues and theireffect on dif-ferent physiological variables. On the other hand, use of this typeof shading net varies the inputs of contaminants relative to the

exterior, and also generates a higher degree of heterogeneity withinthe steel frame(due to e.g. dripping points).


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4.3. Shelter systems

In a numberof cases, when the objective is to quantify only thedry deposition (11% of the studies reviewed), shelters orcovered sites (e.g. belowbalconies; Rivera et al., 2011) are used to

prevent exposure of the moss bags to wet deposition and/or loss byleaching ofsome of the retained contaminants (Couto et al., 2004a;Zechmeister et al., 2006a,b). Several different systems are used tocover the bags, including PVC sheets (Fabure et al., 2010), plasticcontainers such as plant pots (Lodenius, 1998; Sun et al., 2009),and inverted funnels (Xiao et al., 1998).

The use ofcovers to prevent exposure of the moss to wet deposi-tion does not necessarily imply that thedry deposition can be accu-rately quantified. Use of a cover also prevents the largestparticles reaching the moss, and may alter the dynamics ofparticledeposition (Little,1977). Likewise, covers will also alter the form andintensity with which the wind affects the moss samples (Tavaresand Vasconcelos,1996), and thus influence bioconcentration of thecontaminants (e.g.loss ofpreviously retained particles).

Only one study has used high sided sheltering (Amblard-Gross et al., 2002), which allows vertical atmospheric depositiononto the moss from the open circular area around the moss, and

prevents influence on lateral capture from the wind.

4.4. Positioning of moss bags

The way in which the moss bags are positioned will only affect flatbags, as uptake occurs in all directions in spherical bags (Goodmanand Roberts, 1971). Theposition of flat bags mustbe considered in re-lation to the ground (i.e. horizontal or vertical) and the focalpoint ofcontamination. Flat bags are usuallypositioned vertically, which en-able the capture ofcontaminantspresent in the air (gases and small

particles with low sedimentation rates) from the horizontal windflowon to the bags. Horizontal positioning of bags, which is less com-mon (in 33 studies), increases the capture ofcontaminantsby gravi-tational sedimentation and wet deposition (Goodman et al., 1975).The latter requires installation of some type of complex support(see Section 4.5); this is a practicaldisadvantage compared with thevertical arrangement,which usually involves simpler systems.

The orientation of the bags relative to the source ofcontaminationis only important in verticallypositioned bags, and is not mentionedin most studies. The orientation of the bags relative to the source ofcontamination has been taken into account when using transplantsto monitor isolated focal points of contamination. This enables theflat surface of the bag to be directed towards the source being moni-tored(Temple et al., 1981). Gailey and Lloyd (1986b) showedthat re-sults obtained with vertically positioned bags of Sphagnum spp.(which were washed withacid and not oriented exactly perpendicu-lar to the main source of airborne metals) were less replicable thanthose obtained with perpendicularly positioned bags. As withhori- zontally positionedbags, sometype ofsupport mustbe used toorient verticallypositioned bags, which is a practical complication.In light of the above, it is clear that the orientation of vertically

positioned bags in relation to the focal point of contaminationmust be taken into account (i.e. perpendicularto the focal point oranotherspecific orientation).

4.5. Location and type ofsupport used

The moss bags shouldbe placed as far as possible from obstacles(e.g. buildings, vegetation, etc.) that may interfere in theexposure of the moss toatmospheric contaminants. Once the site isselected, the moss bags are suspended (e.g. by a nylon fibre) at acertain height (see Section 4.6) from different structures (e.g.trees, lamp posts, etc.), by use of different types of supports. Thesupports (e.g. plastic tubes, glass fibre poles, etc.) should be madefrom inert materials


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that are not affectedby the contaminants under study, and do not re-lease contaminants during exposure. They should maintain the mossbags separate fromeachotherand as far as possible from structures,inorderto prevent effects such as screening ordripping.Although themoss bags were suspended fromvegetation in 15 of the studies, thisoption should not be used; vegetation is not inert and it may shieldthe moss from atmospheric contaminants and also exert aconcentra-tion effect due to dripping from leaves and twigs onto the bags

(Little and Martin, 1974).In some cases, more complex supports (see Section 4.4) have

been used to suspend moss bags (Tretiach et al., 2007;Makholm and Mladenoff, 2005; Temple et al., 1981). For example,Temple et al. (1981), constructed a special U-shaped support inwhich therectan- gular moss bags are inserted into a sleeve; anotchin the support prevents the holder (and therefore the moss bag)from turning about its vertical axis; this allows the flat surface of thebag to bedirected towards the source being monitored. Tretiachet al. (2007) used two complex types of lattice arrangements toexpose bags on the roof ofautomated air quality monitoring stationslocated in urban areas in Italy; several bags containing differentbiomaterials, including moss, were arranged on the lattices so thatthe bags did not overlap and thesamenumberof bags faced eachcardinal direction.

4.6. Height ofexposure

There is great variability as regards the height ofexposure of themoss bags, ranging from ground level (see e.g. Huttunen etal.,

1981; Samecka-Cymerman and Kempers, 2007) to almost 30 m(Culicov et al., 2005; Rivera et al., 2011). The information is usually

provided (82% of studies) and varies as follows: b 4 m(82%);

47.5 m (9%); 7.511.5 m (4%), and > 11.5 m (5%). Thedifferences in height affect the results because air-flow andturbulence vary enor- mously with height above the ground, whichgreatly influences the amounts of contaminants collected (Adamo et

al., 2011). The latter authors found that at low heights (4 m), thecontamination detected in a narrow street in anurban area of Napleswas related to emissions from traffic and suspended dust fromroads, whereas at higher heights (12 and 20 m), the movement ofair masses favoured accumulation of elements originating fromlong-distance transport and cationsofmarine origin.

Different factors must be taken into considerationwhen selectingthe height of exposure. These include the objectives of the study(Little and Martin, 1974) (e.g. assessment of the quantities ofcontam- inantsinhaledby people from the air) and practical aspects(e.g. the availability of supports from which to suspend the bagsand the need to suspend the bags at a sufficient height to preventloss through vandalism).

4.7. Duration ofexposure

There is also a great deal of variability as regards the period ofexpo-sure ofthe moss bags, and this has been one of most frequently studiedaspects (see e.g. Basile et al., 2009; Gailey and Lloyd, 1986d; Ratcliffe,1975; Tremper et al., 2004; Vasconcelos and Tavares, 1998). Theduration of exposure ranges between 1 week (see e.g. Tavares andVasconcelos,

1996) and 20 months (Evans and Hutchinson, 1996). This informationisprovided in almost all studies (98%) and varies as follows: b 1 month(13%); 1b 2 months (41%); 2b 3 months (23%); 3b 6 months(16%), and >6months (7%).

Ratcliffe (1975)proposed a series ofcriteria in order todeterminethe most appropriate length ofexposure forthe accumulation ofmostcontaminants: (i) detectable accumulated concentrations; (ii) reli-able values (i.e. high replicability); and (iii) an exposure periodwithin the limits ofpractical considerations. The concentrations ofelement in moss should also increasealmost linearly over the expo-

sure period.


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In order to evaluate the above criteria, the moss bags wereex- posed for different periods of time after the same starting

point in several studies (28%). The replicability of resultsgenerally tended to increase with the time of exposure of themoss transplants. For studies in which a minimum of threeexposure periods were tested, the differences in the coefficients ofvariation (CV) of the concentrations of contaminants areshown in Fig. 4. In general terms, irrespective of other factors(e.g. species, pre-exposure treatment, shape of bag, etc.), notrends in the CV were observed after3045 days of exposure, and the variations tended to be lower for

higher concentrations (usually b 20%). The largest fluctuations in theCV were observed for contaminants present at low concentrations(i.e. Ni and Cr; Gailey and Lloyd, 1986d; Fig. 4). However, the mostmarked decrease in the CVs corresponded to the shortest exposureperiods (i.e.3045 days for Cu, Fe, Mn, Pb and Zn; Gailey and Lloyd, 1986d; Fig. 4),reaching below 20% formost elements.

For studies in which a minimum of three exposureperiods wereconsidered, and without taking into account other factors suchas the species etc., we have plotted the rate of uptake ofcontaminants against the time of exposure (Fig. 5). The uptake ofcontaminants by the moss was not linearly related to the exposure

period. Moreover, the rate rarely depended on time (for exceptionssee the following: Cu and Fe in Vasconcelos and Tavares, 1998; Cd

and Mn in Anii et al., 2009a), and the greatest changes wereobserved during the first exposure period. The concentrations ofcontaminants in the moss tissues often increased with theexposure time, although there was sometimes a negativerelationship (e.g. for Cr, Ni, Pb and Zn: Gailey and Lloyd, 1986d).The lack ofa relation between the rate of uptake and the time ofexposure may depend on diverse factors (Anii et al.,2008). Firstly,there may be differences in the atmospheric levels of thecontaminants during the different exposure periods. Secondly, theweather conditions (e.g. wind, rain, etc.) may affect pollutantavailability (e.g. via solubilization) as well as the impact of airborneparticles and their interception, and may also modify the uptake ca-pacity of non-irrigated moss samples (see Sections 2.3.2 and 4.1).For example, during 6-week exposure periods in the same urban en-vironment during spring in twoconsecutive years, the accumulationof elements by H. cupressiforme increased during wetter weatherand when concentrations of airborne particulate matter (PM10)

were higher (Giordano et al., 2009). The latterauthors also observedthat moss bags exposed for a 12-week period accumulatedhigher concentrations of elements than the sum of thoseaccumulated in two consecutive 6-week periods, indicating that inthe study area (with low pollution levels) neither 6- or 12-weekexposure caused saturation of the biomatrix capacity to retainelements.

On the basis of the above findings, we propose use ofan exposureperiod ofbetween 30 and 45 days. Thisensures accumulation of ele-ments and adequate replicability of the results, and is alsofeasible from apractical point of view (and in fact is the mostcommonly used exposure period reported in the literature

consulted).Moreover, when a relationshipbetween the uptake andexposure time exists, the slope of the corresponding regressionline is usually steepest within this period.

4.8. Number ofbags

The numberof moss bags exposed in activebiomonitoring studiesis very variable and rangesbetween 1 and 30. Although replicate bagswereincluded in most studies (n = 23 in 28% of the studies, n= 4in

13% of the studies, n= 5 in 7% of the studies, n= 6b 15 in 9% of thestudies and n=> 15 in 4% of the studies), a large proportion (39%)of the studies did not includereplicates.

One way ofselecting the most suitable number ofmoss bags is to

considerthe level oferror allowed inestimating the mean concentra-tions of contaminants in the moss tissues. If it is assumed thatthe concentrations are normally distributed among bags exposed atthe


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Coe

fficientofvariation(CV)


1.5Al As Cd

1.0

0.5

0.0

1.5Cu Cr Fe

1.0

0.5

0.0

1.5Ni

1.0

0.5

0.0

1.5V Zn

Pb

0 30 60 90 120 150

1.0

0.5

Basile et al. (2008) U

Galey & Lloyd (1986) U

Basile et al. (2009) I

Basile et al. (2009) A

0.00 30 60 90 120 150 0 30 60 90 120 150

Time (days)

Fig. 4. Changes in the coefficients of variation (CV) of the concentrations of contaminants in different studieswith moss bags over time for each metal. The following are

indicated beside each study: the numberofreplicates used in each study, the species used and the area where the study was carried out. In each graph,the level corresponding

to the 20%

error is represented by a dotted line (I = industrial; A= agricultural; U = urban; Scc = Scorpiurumcircinatum; Sg = Sphagnum girgensohnii; SSp = Sphagnum sp.).

same place as replicates (unfortunately this information is not avail-able in the only study in which n=30;Anii et al., 2009a) and themean and standard deviation are known, the number ofreplicate bags that will provide a certain percentage of errorcan be

calculated ([N =(t2

2

) /(D2

2

)], where 2

and are the varianceand mean of the data; D is the level of errorassumed and t in thevalue of the sta- tistics in a Student's t test at the 5% significancelevel). The number of bags required for errors of 10, 15 and 20%(calculated from data available in the literature), for exposure

periods of 30 and 3060 days (chosen on the basis of theinformation in Section 4.7) is shown in Tables 1 and 2respectively. In some cases the number of bags required isextremely high (much higher than 50: i.e. n > 4000 for an error of10%) because of the large difference in the concentrations in the

bags exposed together. For some elements, such as Cr, Hg and Pb,several replicate bags (i.e. n > 10) are required forboth periods,unlike for other elements such as Al, Fe, and Mn. In generalterms, the concentrations ofelements in the moss bags exposed for

periods of3060 days were less variable than in bags exposed for 30days, as indicated by the modal values shown in both tables. Themaximum modal numbers of bags for allelements,corresponding tosampling

errors of 10, 15 and 20%, were 7, 3 and 2 for a 30 dayexposure period,and 3, 3 and 2 for an exposure period of 3060 days, respectively.

4.9. Initial and control concentrations

In most studies (64%), part of the material destined for use in themoss bags is conserved to determine the initial concentrations ofeach of the contaminants under study. These concentrations arethen used to calculate enrichment factors (EF =finalconcentration /initial concentration) and the netenrichment (NE= final concentra-tion minus initialconcentration) for each of the transplants and con-taminants studied.

It is also important to apply certain measures toprevent contam-ination of the samples outside theexposure period or exposure site.Gailey and Lloyd (1986b) found that the replicability of the resultsdecreased greatly when strict guidelines for sample handling werenot followed, and therefore thathandling ofsamples during prepara-tion, collecting,transport to the laboratory and preparation for analy-

sis can cause different degrees of contamination or loss of adheringparticles. Some studies includedcontrolbags, which were subjected


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Rateofelementuptake(ggday-)


200Al

0.06As

0.04Cd

1500.04

0.03

100

500.02

0.02

0.01

0

2.5Cu

2.0

1.5

1.0

0.5

0

1.5Cr

0.8

0

-0.8

0

350Fe

200

50

0 -1.5

60Mn

0.9Ni

400.4

-100

5.5

3.5

Pb(a)

20 -0.1 1.5

0 -0.6 -0.5

-20 -1.1 -2.5

25 Pb (b)0.9

V15

Zn

1019

0.6

135

0.3

7 0

-5 0 -50 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150

Time (days)

Basile et al. (2008) U; n=3; Scc

et al.Basile et al. (2009) A; n=3; Scc

Vasconcelos & Tavares (1998) U; n=11; Sau

Ratcliffe (1975) I 3; n=?;Spa y Ss

Ratcliffe (1975) I 4; n=?; Spa y Ss

Galey & Lloyd (1986d) U; n=10; SSp Ratcliffe (1975) I 1; n=?; Spa y Ss Tavares & Vasconcelos (1996) A; n=?; Sau

Basile et al. (2009) I; n=3; Scc Ratcliffe (1975) I 2; n=?; Spa y Ss Tavares & Vasconcelos (1996) C; n=?; Sau

Fig. 5. Changes in the rate of uptake (g g day

) of different contaminants in different studies with moss bags. The following are indicated beside each study: the area where

the study was carried out, the number ofreplicates used in each study and the species used. As numerous studies determined the concentrations ofPb, the data are included in

two graphs (U = urban; I = industrial; A = agricultural; C = control; Sau = Sphagnum auriculatum; Scc = Scorpiurum circinatum; Sg= Sphagnum girgensohnii; Spa =Sphagnumpap-

illosum; Ss = Sphagnum subsecundum; SSp = Sphagnum sp.).

to all handling and transportation steps,but which were not exposedin the study area (Ares et al., 2011; Coutoet al., 2004b). In one study,samples of c. 700 mg ofdifferent biological materials were collected;200 mg were used to determine the initial concentration, and theremaining 500 mg were placed in the bag for exposure. For each bio-material, 12 bags were prepared and analyzed to assess thevariability in the initial concentrations, which, for most of theelements, was lowest in water-washed moss and highest in acid-washed moss (Giordano et al., 2009).

5. Post exposuretreatments

After exposure, the samples were sometimes washed in thelabora- tory prior to being prepared for analysis (in18% of the studiesreviewed), with washing times that range from a few seconds(Samecka-Cymerman

and Kempers, 2007) to 10 min (see e.g. Fernndez et al., 2000). The aimof this washing step is to remove weaklybound elements or elements

bound on particles deposited on the surface of the moss, and alsoto evaluate the effect of rainfall events during exposure. Furtherresearch is required to optimize the post exposurewashing procedurefor dead moss, by determining how effective the procedure is andwhether it alters theextracellularequilibrium.

As regards the use of live moss, some authors (Wells and Brown,1990) have indicated that washing times of more than 30 s mayalterthe extracellular equilibrium, thuscausing loss of at least partof the contaminantsbound to the cell wall and the external face ofthemembrane. Therefore, long washing times are notrecommended.

On the other hand, Aboal et al. (2011)have recently demonstrated,by use of scanning electron microscopy, that in samples of nativeP. purum, washing for 30 s is ineffective at removing the

particles.


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A.Aresetal./Science

ofthe

Tota

lEnvironment432

(2012)14

3

158

155

Table2

Calculation of the numberof replicates required for errors of 10, 15 and 20% respectively, for different studies considering various contaminants and an exposure period of 3060 days. An asterisk indicates n > 50, and indicates that

thee modal value could not be calculated. (L= livemoss; F= flat moss bag; Ps = Pleurozium schreberi; Pp= Pseudoscleropodium purum; Ep= Eurhynchium praelongum; Up= Unpolluted; Sg =Sphagnum girgensohnii; U= Urban; I=

Industrial; S = spherical moss bag; Scc = Scorpiurum circinatum; R= Rural; D = devitalized moss; Hc= Hypnum cupressiforme; Am = acid washed moss; C = Control; Sp = Sphagnum palustre; SSp = Sphagnum sp.; Hs =

Hylocomium splendens; Sca = Sphagnum capillifolium; St= Sphagnum teres; T= Traffic).

References Details Al As Ba Be Cd Co Cr Cu Fe Hg Mn Ni Pb Se Sr Zn

Amblard-Gross et al.(2002) L, F, Ps, Pp, Ep, Up 1, 1, 1 1, 1, 1 1,1,1 1,1,1Anii et al. (2009b) L, F, Sg, U 15, 7, 4 *, 27,15 18, 8,4 15, 7,4 7, 3, 2 24, 11,6 32, 14,8 21, 10,5 7, 3,2 9, 4, 2 22, 10,6 5, 2,1 13, 6,3 9, 4, 2

Ares et al. (2011) L, F, Pp, U-I *, 32,18 *, 49,28 19, 9,5 *, *, 32 *, *,30Basile et al. (2008) L, S, Scc, U 2, 1, 1 2, 1, 1 1, 1, 1 3, 1, 1 2, 1, 1 1, 1, 1 1, 1,1 1, 1, 1 1, 1, 1

Basile et al. (2009) L, S, Scc, I 8, 3, 2 6, 3, 2 2, 1, 1 5, 2, 1 1, 1, 1 1, 1, 1 1, 1,1 1, 1, 1 3, 1, 1

L, S, Scc, R 2, 1, 1 2, 1, 1 3, 1, 1 3, 1, 1 2, 1, 1 1, 1, 1 1, 1,1 2, 1, 1 1, 1, 1

Castello (1996) L-D, S, Hc, I 1, 1, 1 26, 11,6 30, 13,7 20, 9, 5 1, 1, 1 *, *, * 15, 7, 4 23, 10,6

L-D, S, Hc, I, Am 12, 5, 3 38, 17,10 42, 19,11 11, 5, 3 15, 7, 4 8, 4,2 37, 16,9 18, 8, 5

L-D, S, Hc,Up 8, 3, 2 13, 6, 3 *, *, 30 31, 14,8 12, 5, 3 14, 6,4 42, 19,11 *, 26,14

L-D, S, Hc, C, Am 25, 11,6 *, 46,26 30, 13,8 *, 32,18 8, 4, 2 *, *, 33 *, *, 32 *, *,31

Castello (2007) L-D, S, Hc, I *, *, * *, 34,19 *, *, * *, *, * *, *, * *, *, * *, *, 43 *, *, * *, *, * *, *, 31

L-D, S, Pp, I 23, 10,6 *, *, * *, *, * *, *, * *, *, * *, *, * *, *, 41 *, *, * *, *, * *, *, 36

Fernndez andCarballeira (2000) L, F, Pp, I *, 34,19 *, *, 37 29, 13,7 *, *, * *, *, * 30, 13,7

Fernndez et al. (2000) L, F, Pp, I

Gailey and Lloyd(1986d) D, S, Sp, U-I,Am *, *, * *, *, * *, *, 34 *, *, * *, *, * *, *, * *, *, *

Gailey and Lloyd(1986d) D, S, SSp,U-I,Am 2, 1, 1 22, 10,6 3, 1, 1

Gailey and Lloyd(1986b) L, S-F, SSp,U-I 1, 1, 1 1, 1, 1 1, 1, 1 4, 2,1 41, 18,10 1, 1, 1 1, 1, 1Little and Martin(1974) L, S, SSp, I

Lodenius (1998) D, S, SSp, I, Am *, *, *

Rivera et al. (2011) L, F, Hs, U *, *, * *, *, * *, 43,24 *, *, 39 *, *, * *, *, * 31, 14,8

Yurukova and Ganeva(1997) L, F, Sca, Up 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1L, F, St, Up 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1

L, F, Sca, I 1, 1, 1 19, 9, 5 7, 3, 2 1, 1, 1 5, 2, 1 1, 1, 1 1, 1,1 2, 1, 1 2, 1, 1 6, 3,2 3, 2, 1L, F, St, I 1, 1, 1 26, 12,7 8, 4, 2 1, 1, 1 6, 3, 1 1, 1, 1 2, 1,1 11, 4, 2 2, 1, 1 8, 3,2 3, 1, 1

Zechmeister et al.(2006b) L, F, Hs, T

Modal value 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 3, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 1, 1, 1 3, 3,2 1, 1, 1


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Table 3

Variables in the methodology of the mossbag technique used to monitor the air quality, and studies carried outwith the aim of optimizing these variables, the options most

commonly used, and finally therecommendations for a harmonised protocol.

Variable Optimization Option most commonly used Recommendations

1. Preparation of the moss

1.1. Selection of species Needs further research Sphagnum sp. (51%) Clonedspecies

1.2. Selection of material Needs further research Apical portion (19%) Greenapical portions (5 cm)

1.3. Pre-exposure treatments and vital state Devitalized Live moss (63%) Devitalized

1.3.1. Washing with cellularextractants Devitalized

1.3.1.1. Number of washes 1 wash with EDTA (10mM) + 1with dimercaprol (30 mM)

1.3.1.2. Duration of washing 20min

1.3.1.3. Shaking Wash with shaking

1.3.1.4. Weight of moss per volume of extractant 1 L solution per 200 g dw

1.3.2. Washing with water Needs further research Washedwith water(72%) Water washed

1.3.2.1. Number of washes 3 times (47%) 3times

1.3.2.2. Duration of washing 20min

1.3.2.3. Shaking Wash with shaking

1.3.2.4. Type of water Distilled water (80%) Distilled water

1.3.2.5. Weight of moss per volume of water 10 L of water per 100 g dw 10 L water per 100 gdw

1.3.3. Devitalizing treatment Oven drying, 24 h 120 Ca,b

Acid washed (25%) Oven drying, 24 h 120 C

2. Preparation of transplants

2.1. Mesh material Inert material Nylon mesh (71%) Nylon mesh

2.2. Mesh size Large without loss of material 12 mm (35%) 2 mm

2.3. Shape of bag Sphericalc

Spherical moss bags (35%) Spherical (ensuringsinglelayerof moss)

2.4. Size of bag 30 mg cm2d

b 40 mg cm2

(32%), 30 mg cm2

3. Exposure

3.1. Shading system General heterogeneity no No conclusion

3.2. Cover Underestimation of deposition no No conclusion

3.3. location and type of support used Free of obstacles,suspended from inert

support by nylonthread

3.4. Height of exposure Needs further research b 4 m (82%) 4 m

3.5. Duration of exposure Between 30 and 60 days 30b 60 days (41%) 45 days

3.6. Numberofbags for site 1 (39%) 3 bags

3.7. Initial concentrations and controls 3 controls and 3 initialtimes

4. Post exposure treatment No treatment No treatment (72%) No treatment

aFernndez et al. (2010).

bGiordano et al. (2009).

cGailey and Lloyd (1986d).

dTemple et al. (1981).

achieve anotherof the proposed objectives, i.e. a linearrelationship be-tween the concentrations of the elements captured by the moss andthe concentration in the atmosphere. This is often required ofbio-monitors so that their use can be considered as a valid alternative tocon- ventional monitoring techniques. However, moss bags do notact as integrators of contaminants and often show negative rates ofcontami- nant uptake. The content ofelements in the mosssamples isthe result ofadditive uptake and also of loss ofaccumulated elementsby leaching, and therefore thesamples cannot exhibit linear relationshipswith levels of atmospheric deposition. Therefore biomonitoring withmoss samples should be used in conjunction with conventionalmonitoring. Used together, these techniques will provide aninexpensive, flexible and dense monitoring design able to indicate

spatial and temporal trends and vertical and horizontal gradients foravariety ofinorganic and organ- ic contaminants.

The application of a standardized protocol for the use of moss bagsshould not overlook the ways in which the technique works.Stan- dardization should providemore comparable results, but it doesnot necessarilyproduce a better understanding of the biologicalprocess- es and mechanisms underlying the accumulation ofcontaminants by moss in bags. In this sense, scientists shouldcontinue to investigate the value and limits ofbiomonitoring.

Acknowledgements

The present study was fundedby the SpanishMinistry of Science,

project CTM2011-30305 andPrograma Nacional FPU.

References

Aboal JR, Couto JA, Fernndez JA, Carballeira A. Definition and number of subsamplesfor usingmosses as biomonitors of airborne traceelements. Arch Environ ContamToxicol2006;50:8896.

Aboal JR, Couto JA, Fernndez JA, Carballeira A.Physiological responses to atmosphericfluorine pollution in transplants of Pseudoscleropodium purum. Environ Pollut2008;153:6029.

Aboal JR, Prez-Llamazares A, Carballeira A, Giordano S, Fernndez JA. Shouldmoss samples used as biomonitors of atmospheric contamination be washed?Atmos Environ 2011;45:683740.

Acar O. Biomonitoring and annual variability of heavy metal concentration changesusing moss(HypnumcupressiformeL. ex. Hedw.) in Canakkaleprovince.J Biol Sci

2006;6:3844. Adamo P, Giordano S,Vingiani S, Cobianchi RC, Violante P. Trace elementaccumulation by moss and lichen exposed in bags in the city ofNaples (Italy).

Environ Pollut

2003;122:91-103.Adamo P, Crisafulli P, Giordano S, Minganti V, Modenesi P, Monaci F, et al. Lichen and

mossbags as monitoring devices in urban areas. Part II: trace element content inliving and dead biomonitors and comparison with synthetic materials. EnvironPollut 2007;146:3929.

Adamo P, Bargagli R, Giordano S, Modenesi P, Monaci F, Pittao E, et al. Naturaland pre-treatments induced variability in the chemical composition andmorphology of lichens and mosses selected for active monitoring of airborneelements. Environ Pollut2008a;152:119.

Adamo P, Giordano S,Naimo D, Bargagli R. Geochemical properties of airborne partic-ulatematter (PM10) collected by automatic device andbiomonitors in a Mediter-ranean urbanenvironment. Atmos Environ 2008b;42:34657.

Adamo P, Giordano S, Sforza A, Bargagli R. Implementation of airborne trace elementmonitoring with devitalised transplants of Hypnum cupressiforme Hedw.:assessment of temporal trends and element contribution by vehicular traffic inNaples city. Environ Pollut2011;159:16208.

Al-Radady AS, Davies BE, French MJ. A new design of moss bag to monitor metaldeposition bothindoors and outdoors. Sci Total Environ1993;133:27583.

Amblard-Gross G, FerardJF, Carrot F, Bonnin-Mosbah M,Maul S, Ducruet JM, et al. Biolog-

ical fluxes conversion and SXRF experiment with anew active biomonitoring toolforatmosphericmetals and trace element deposition. EnvironPollut2002;120:4758.


23/25


Anii M, Tomaevi M, Tasi M, Raji S, Popovi A, Frontasyeva MV, et al.Monitoring of trace element atmospheric deposition using dry and wet moss

bags: accumulation capacity versus exposure time. J Hazard Mater2009a;171:1828.

Anii M, Tasi M, Frontasyeva MV, Tomaevi M, Raji S, Strelkova LP, et al.Active biomonitoring with wet and dry moss: a case study in an urbanarea. Environ Chem Lett2009b;7:5560.

Archibold OW, Crisp PT. The distribution of airbornemetals in the Illawarra region ofNew South Wales, Australia. Appl Geogr1983;3:33144.

Archibold OW. The metal content of wind-blown dust from uranium tailings innorth- ernSaskatchewan. WaterAir Soil Pollut1985;24:6376.

Ares , Fernndez AJ, Aboal JR, Carballeira A. Study of the air quality in industrialareas of Santa Cruz de Tenerife (Spain) by active biomonitoring withPseudoscleropodium purum. Ecotoxicol Environ Saf2011;74:53341.

Bargagli R. Trace elements in terrestrial plants. Berlin:Springer; 1998.Bargagli R, Brown DH, Nelli L. Metal biomonitoring with mosses: procedures for cor-

recting for soilcontamination. Environ Pollut 1995;89:16975.Basile A, Sorbo S, Aprile G, Conte B, Cobianchi RC.Comparison of the heavy metal bio-

accumulation capacity of an epiphytic moss and anepiphytic lichen. EnvironPollut

2008;151:4017.

Basile A, Sorbo S, Aprile G, Conte B, Cobianchi RC, Pisani T, et al. Heavy metaldeposition in theItaliantriangle of death determinedwith themoss Scorpiurumcircinatum. Environ Pollut2009;157:225560.

Berg T, Steinnes E. Recent trends in atmosphericdeposition of trace elements in Nor-way as evidentfrom the 1995 moss survey. Sci Total Environ1997;208:197206.

Boquete MT, Fernndez JA,Aboal JR, Carballeira A. Analysis of temporalvariability inthe concentrations of some elements in the terrestrial moss Pseudoscleropodium

purum. Environ Exp Bot 2011a;72:2106.Boquete MT, Fernndez JA, Aboal JR, Carballeira A. Are terrestrial mosses good

biomonitors of atmospheric deposition of Mn? Atmos Environ 2011b;45:270410.

Brown DH, Brown RM. Mineral cycling and lichens: thephysiological basis. Lichenologist1991;23:293307.

Brown DH, Wells JM. Sequential elution technique for determining the cellularlocation of cations. In: Glime JM, editor. Methods in BryologyProc. Bryol.Meth.Workshop, Mainz, Hattori Bot. Lab.,Nichinan; 1988.p. 22733.

Cameron AJ,Nickless G. Use of msses as collectors of airborne heavy metals neara smelting complex.WaterAir Soil Pollut 1977;7:11725.

Carballeira CB, Aboal JR, Fernndez JA, Carballeira A. Comparison of the accumulationof elements intwo terrestrial moss species. Atmos Environ2008;42:490417.

Carpi A, Weinstein LH, Ditz DW. Bioaccumulation ofmercury by Sphagnum moss neara

municipal solid waste incinerator. J Air Waste Manage Assoc 1994;44:66972.Castello M. Monitoring of airborne metal pollution by moss bags: a

methodological

study. Stud. Geobotanica 1996;15:91-103.

Castello M. A comparison between two moss speciesused as transplants forairborne trace element biomonitor ing in NE Italy. Environ Monit Assess

2007;133:26776. eburnis D, Valiulis D.Investigation of absolute metal uptakeefficiencyfromprecipita-

tion in moss. Sci Total Environ 1999;226:24753.Clough WS. Deposition ofparticles on moss and grasssurfaces. Atmos Environ 1975;9:

11139.

Couto JA, Fernndez JA, Aboal JR, Carballeira A. Activebiomonitoring of elementuptake with terrestrialmosses: a comparison ofbulk and dry deposition. Sci TotalEnviron

2004a;324:21122.

Couto JA, Aboal JR, Fernndez JA, Carballeira A. A newmethod for testing the sensitivity ofactive biomonitoring: an example of its application to a terrestrial moss.Chemosphere

2004b;57:3038.

Culicov OA, Yurukova L. Comparison of element accumulation of different moss- andlichen-bags,exposed in the city of Sofia (Bulgaria). J AtmosChem 2006;55:1-12.

Culicov OA, Mocanu R, Frontasyeva MV, Yurukova L, Steinnes E. Active mossbio- monitoring applied to an industrial site in Romania: relativeaccumulation of 36 elements inmoss-bags. Environ Monit Assess2005;108:229

40.Decker EL, Reski R. The moss bioreactor. Curr OpinPlant Biol 2004;7:16670.Decker EL, Reski R. Moss bioreactors producing improved biopharmaceuticals.

Curr

Opin Biotechnol 2007;18:3938.

Decker EL, Reski R. Current achievements in the production of complex bio-pharmaceutica lswith moss bioreactor. Bioprocess Biosyst Eng2008;31:39.

Dmuchowski W, Bytnerowicz A. Long-term (19922004)record of lead, cadmium, andzinc air contamination in Warsaw, Poland:determination by chemical analysis ofmoss bagsand leaves of Crimean linden. Environ Pollut2009:341321.

European Parliament Directive 2008/50/EC of the European Parliament and ofthe

Council of 21 May 2008 on ambient air quality andcleanerair for Europe. OfficialJournal,1e44. 11.062008, L152.

Evans CA, Hutchinson TC. Mercury accumulation in transplanted moss and lichens athigh elevationsites in Quebec. Water Air Soil Pollut1996;90:47588.

Fabure J, MeyerC, DenayerF, Gaudry A, Gilbert D,BernardN. Accumulation capacitiesofparticulatematterin an acrocarpous and apleurocarpous moss exposed atthree differentlypollutedsites (industrial, urban and rural). WaterAirSoil Pollut

2010;212:20517.Fernndez JA, Carballeira A. Differences in the responses of native and transplanted

mosses to atmospheric pollution: a possible role of selenium. Environ Pollut2000;110:738.

Fernndez JA, Ares A, Rey-Asensio A, Carballeira A,Aboal JR. Effect of growth on activebiomonitoringwith terrestrial mosses. J Atmos Chem2010;63:1-11.

Fernndez JA, Aboal JR, Carballeira A. Use of native and transplanted mosses ascomple- mentary techniques for biomonitoring mercury around an industrialfacility. Sci Total Environ2000;256:15161.

Fernndez JA, Aboal JR, Couto JA, Carballeira A. Sampling optimization atthe sampling-site scale for monitoring atmospheric deposition using mosschemistry. Atmos Environ2002;36:116372.


24/25

Gailey FAY, Lloyd OL. Atmospheric metal pollutionmonitored by spherical moss bags:a case study ofArmadale. Environ Health Perspect1986a;68:18796.

Gailey FAY, Lloyd OL. Methodological investigations intolow technology monitoring

monitoreo con musgo

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