Ei

KD

a

ARRA

KBAEBS

1

tc((tgAtsLtgcoc

r(

1h

Ecological Indicators 36 (2014) 392– 399

Contents lists available at ScienceDirect

Ecological Indicators

jou rn al hom epage: www.elsev ier .com/ locate /eco l ind

valuating relationships between mercury concentrations in air andn Spanish moss (Tillandsia usneoides L.)

athryn T. Sutton ∗, Risa A. Cohen, Stephen P. Vivesepartment of Biology, Georgia Southern University, P.O. Box 8042, Statesboro, GA 30460, USA

r t i c l e i n f o

rticle history:eceived 13 March 2013eceived in revised form 15 July 2013ccepted 5 August 2013

eywords:ioindicatortmospheric pollutantpiphyteromeliadoutheastern United States

a b s t r a c t

Measurement of mercury vapor is essential given that it is transported globally, and once depositedcan be converted to methylmercury, a dangerous neurotoxin. A study was conducted in southeasternGeorgia and northern Florida, USA to determine whether Spanish moss, an epiphytic vascular plant com-mon to the southeastern United States, has the ability to retain mercury in its tissues over time, andto detect atmospheric mercury at relatively low concentrations from nonpoint sources. Spanish mossplants exposed to 10× and 100× ambient concentrations of mercury vapor increased tissue concentra-tions by 13.7 ± 11% and 74.1 ± 17% respectively, and then retained the mercury over two weeks followingremoval from the source. There was a strong trend of increasing Spanish moss mercury concentrationswith increasing air concentration in resident populations across urban, rural inland, coastal and indus-trial sites. Transplanted Spanish moss around an industrial site contaminated with mercury exhibited a

164.8 ± 8.7% increase in mercury concentration after two weeks. Mercury concentration of Spanish mosstransplanted to rural inland sites also increased after two weeks, while the change in transplant concen-tration at coastal sites was more variable. This study shows that Spanish moss possesses characteristicsimportant for use as a bioindicator of atmospheric mercury, and can potentially be adapted as a tool forobtaining time-integrated atmospheric mercury data to add to existing atmospheric mercury monitoringprograms.

. Introduction

Comprehensive measurement of atmospheric mercury is essen-ial given that its deposition can lead to methylation, and thereforeonversion to a toxin that can bioconcentrate in food websWagemann et al., 1995), be stored in sediments and biotaCanário et al., 2007; Williams et al., 1994) or be re-released tohe air through transpiration by plants or volatilization of theaseous form, Hg0 (Lindberg et al., 2002; O’Driscoll et al., 2007).tmospheric mercury concentrations and deposition patterns are

ypically estimated using mechanical methods, such as gold trapystems (Calasans and Malm, 1997), and the Differential Absorptionidar technique which measures atmospheric mercury concentra-ions along a laser beam (Grönlund et al., 2005). However, obtainingaseous atmospheric mercury measurements that are spatially

omprehensive and temporally integrated using the above meth-ds requires many replicate samples, which can be laborious andostly (Carballeira and Fernández, 2002; Figueiredo et al., 2007).

∗ Corresponding author. Tel.: +1 912 478 1228; fax: +1 912 478 0845.E-mail addresses: ks02695@georgiasouthern.edu (K.T. Sutton),

cohen@georgiasouthern.edu (R.A. Cohen), svives@georgiasouthern.eduS.P. Vives).

470-160X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.ecolind.2013.08.011

© 2013 Elsevier Ltd. All rights reserved.

A potential solution is the use of bioindicators, which offer anintegrative method to estimate atmospheric concentrations anddeposition patterns (De Temmerman et al., 2004).

Plants are often used as bioindicators of heavy metal air pollu-tion (e.g. Brighigna et al., 1997; Davis et al., 2007; Figueiredo et al.,2004; Steinnes et al., 2003) largely because they have a high capac-ity to accumulate heavy metals into their tissue over long timeintervals (Alves et al., 2008; Calasans and Malm, 1997). A plantthat is an effective bioindicator of atmospheric heavy metals suchas mercury must not only be able to take up metal pollutants butalso retain pollutants in tissue for a period of time necessary fortissue concentrations to be representative of average atmosphericconcentrations (Baker, 1981; Falla et al., 2000). In addition, plantswith a wide geographical distribution are effective in ascertain-ing impacts of atmospheric pollution over large spatial scales. Forexample, lichens have been used to assess mercury contaminationsurrounding specific sources, such as a chlor alkali plant (Xantho-ria parietina; Grangeon et al., 2012), as well as across the Pradedand Glacensis ecoregions in Poland and the Czech Republic (Hyp-ogymnia physodes; Kłos et al., 2012). Mosses (true bryophytes) may

also be used as bioindicators of atmospheric heavy metal pollution;Harmens et al. (2010) sampled moss species throughout Europefrom 1990 to 2005, and found lower tissue metal concentrationscoincident with the decline in heavy metal emissions during that

al Indi

tsaa

i(lscraaf2aohame

tdaiap1tahw1oMtf

tsstavt1anbo

mr(iagsmbecoRm

K.T. Sutton et al. / Ecologic

ime period. Furthermore, Suchara and Sucharová (2008) reportedimilar mercury concentrations in the moss, Hypnum cupressiforme,s well as humus, oak and pine barks around a contaminated chlor-lkali plant, suggesting utility as bioindicators of mercury levels.

Spanish moss (Tillandsia usneoides L.), an epiphytic bromeliad,s a potential bioindicator of atmospheric mercury concentrationFigueiredo et al., 2007; Husk et al., 2004). Spanish moss accumu-ates heavy metals from the environment in its tissue due to highurface area, the absence of a cuticle, and high cation exchangeapacity (Alves et al., 2008; Calasans and Malm, 1997). In addition,oots of this bromeliad are virtually non-existent or are used onlys holdfasts, meaning nutrients, water, and atmospheric pollutantsre absorbed directly from the air through scales on the plant sur-ace (Alves et al., 2008; Amado Filho et al., 2002; Wannaz et al.,006). Therefore, pollutants measured in tissue may be related totmospheric sources without complication due to uptake from soilr other media (Carballeira and Fernández, 2002). This plant alsoas a low sensitivity to heavy metal exposure, likely because metalsre retained in its outer scales and not translocated to internalesophyll fibers (Alves et al., 2008; Amado Filho et al., 2002; Bastos

t al., 2004; Figueiredo et al., 2007).Another advantage is that Spanish moss can be easily

ransplanted with little mortality, which allows for estimatingeposition patterns in areas where no suitable bioindicator plantsre present, and establishing baseline measurements of depositionn areas where there is no historical data (Falla et al., 2000). Calasansnd Malm (1997) transplanted Spanish moss inside a chlor-alkalilant with air mercury levels between 1 and 64 �g m−3, and after5 days found tissue mercury increased to 13,500 times greaterhan control plants. In addition, transplanted Spanish moss insidend around gold shops reached 26 ppm, which was 300 timesigher than control plants, and plant concentrations decreasedith increasing distance, up to 20 m from the shops (Malm et al.,

998). Although Spanish moss accumulates mercury either indoorsr in very close proximity to emission point sources (Calasans andalm, 1997; Malm et al., 1998), it is unclear whether Spanish moss

akes up mercury in proportion to atmospheric concentrationsrom nonpoint emissions sources over large geographic areas.

It is also unknown whether Spanish moss retains mercury inissue after exposure to variable atmospheric concentrations. Thetudies by Calasans and Malm (1997) and Malm et al. (1998)howed Spanish moss transplants take up mercury when exposedo a highly elevated and constant source of emission. However,tmospheric mercury concentrations in other areas are typicallyariable due to sporadic emissions and changing wind and precipi-ation patterns (Conti and Cecchetti, 2001; Schroeder and Munthe,998). Therefore, if Spanish moss loses tissue mercury taken upfter intermittent changes in atmospheric concentration, it wouldot be representative of average air concentrations. Mercury muste retained in tissue to effectively use Spanish moss as a biomonitorf atmospheric mercury concentrations.

The purpose of this study was to determine the utility of Spanishoss as an indicator of atmospheric concentrations of mercury in a

ange likely to be encountered in the field. We hypothesized that:1) mercury taken up by Spanish moss is retained in tissue follow-ng uptake; and (2) mercury concentrations in Spanish moss tissuere associated with atmospheric concentrations over a wide geo-raphic range. To test our hypotheses, we conducted field studies inoutheastern Georgia and northern Florida, USA. We predicted thatercury taken up by Spanish moss from the atmosphere would

e retained in tissue regardless of exposure concentration, andxpected air mercury concentrations to not only differ geographi-

ally due to nonpoint emission sources, but also that concentrationsf mercury in Spanish moss tissue would reflect these differences.esults of this study are used to address the capabilities of Spanishoss for the temporal integration of atmospheric mercury from

cators 36 (2014) 392– 399 393

point and nonpoint sources, and its utility as a tool to augmentexisting atmospheric mercury monitoring protocols.

2. Materials and methods

2.1. Retention experiment

To determine how long mercury is retained following uptakeinto Spanish moss tissue, plants with high tissue concentrationsof mercury were allowed to depurate in a low mercury environ-ment. First, plant segments were collected haphazardly at the SaltMarsh Ecosystem Research Facility (SERF) at Skidaway Island, GA,USA (31◦55′39′′ N, 81◦2′33′′ W) from an established populationwith low background tissue mercury levels (0.05–0.16 �g Hg g−1

tissue). Segments were 10–20 cm of new growth from distal endsof each plant at least 1 m from the ground to ensure plants did nothave contact with soil. In the laboratory, plants were washed withmercury-free tap water to remove any particulate mercury on theplant surface. Segments were then mixed together so individualreplicates would be selected at random, and a subsample (n = 6)was analyzed for background mercury concentration.

Next, Spanish moss segments (total biomass, 200 g) were placedinto each of two glass containers and onto latticed cardboard overa pool of volatilizing liquid elemental mercury for one week. Twotreatment groups were obtained by exposing Spanish moss to vaporconcentrations resulting in tissue mercury concentrations of either10× (1.3 ± 0.09 �g g−1) or 100× (12.2 ± 0.26 �g g−1) that of ambi-ent Spanish moss concentrations (0.13 ± 0.003 �g g−1). Individualsegments of Spanish moss from the two treatment groups werehaphazardly tied to individual trees at least 1 m apart along a 100 mtransect in the forest at the SERF site. At 2, 7, and 14 days, 5 seg-ments from each treatment were collected and analyzed for tissuemercury concentration.

Spanish moss tissue mercury concentration data (in this and allsubsequent experiments) were tested for equality of variance usingLevene’s test and for normality using the Shapiro–Wilk W test. Datanot meeting parametric assumptions were either log transformedor analyzed with nonparametric tests. To determine whether Span-ish moss tissue retained mercury and whether responses wereconcentration-dependent, ANCOVA was conducted following logtransformation on treatment group (starting tissue mercury con-centration) with sampling time as the covariate.

2.2. Resident population study

A field study was conducted to examine the relationshipbetween mercury concentration in resident Spanish moss popu-lations and air mercury concentration. Spanish moss fromestablished populations was collected in southeastern Georgia andnorthern Florida, USA between March and September 2011 fromsites categorized as urban, coastal, inland, and industrial (Fig. 1).The urban sites of Jacksonville, FL and Savannah, GA were expectedto have high mercury levels in air and in Spanish moss becausethese are urban centers with large human populations (>500 peo-ple km−2; US Census, 2011). The LCP Chemicals EPA Superfund Sitein Brunswick, GA was also predicted to have high mercury levelsbecause the site has a history of mercury pollution in soils andsediments from industrial activity (Gardner et al., 1978; US EPA,2011; Windom et al., 1976; Winger et al., 1993). In contrast, Georgiacoastal barrier islands (Cumberland Island, Ossabaw Island, andSapelo Island) were predicted to have lower mercury concentra-

tions than urban and industrial sites due to limited public access,a relative lack of development, and location >30 km from urbancenters. However, because the barrier islands are located east ofurban centers, it is possible that prevailing winds could transport

394 K.T. Sutton et al. / Ecological Indi

Fig. 1. Field sites in southeastern Georgia (Magnolia Springs state park, MS; GeorgeL. Smith state park, GLS; Private Land, PL; Savannah, SV; Ossabaw Island, OI; SapeloI(i

mSLfsednon2Msgstmf

2

mwsfS

2

pBhiphot1l

sland, SI; Cumberland Island, CI; Brunswick LCP site, BW) and northern FloridaJacksonville, JX) used in resident population and coastal vs. inland transplant stud-es.

ercury from urban and industrial sites to the coast. Therefore,panish moss was also sampled from Magnolia Springs and George. Smith state parks, which are located west of (therefore upwindrom) urban and industrial sites. The state parks are also ruralites that also have limited infrastructure and public access. Atach field site, three air samples were collected (following protocolescribed in Section 2.3.3). In addition, 20 strands of Spanish mossew growth (2.5 g) were collected haphazardly from distal endsf plants, each from an individual tree, and placed immediately initric acid, fixing mercury in solution. Strands were between 10 and0 cm, which, based on slow maximum growth rates calculated byartin et al. (1981) (∼270 �m day−1 number of leaves−1) repre-

ented new plant growth exposed to the atmosphere within therowing season. Tissue mercury concentrations of Spanish moss atites with differing human influence (urban, coastal, inland, indus-rial) were measured in the laboratory within 48 h of collection and

ean tissue concentrations were correlated to air concentrationsor each site using the Pearson correlation coefficient.

.3. Transplant studies

Spatial patterns of atmospheric mercury uptake by Spanishoss were assessed via transplant studies, conducted at a siteith mercury contamination and at sites without direct emission

ources. All Spanish moss plants were collected for transplantingrom the SERF site following the collection protocol described inection 2.1.

.3.1. Transplants to a mercury-contaminated siteSpanish moss with low background levels of mercury was trans-

lanted to the vicinity of the LCP Chemicals EPA Superfund site inrunswick, GA, a 200–250 ha area consisting of mostly salt marshabitat, in which industrial activities have operated since 1919,

ncluding an oil refinery, a paint manufacturing company, a powerlant, and a chlor-alkali plant (US EPA, 2011). These operationsave contaminated the soils and groundwater with PCBs, volatile

rganics, and mercury (US EPA, 2011). Mercury was dischargedo soils and sediments from the chlor-alkali plant at a rate of

kg Hg day−1 for six years until 1972, resulting in high mercuryevels (up to 27 ppm) in the marsh sediments surrounding the site

cators 36 (2014) 392– 399

(Gardner et al., 1978; Windom et al., 1976). Although 25,000 tonsof contaminated soils were removed in 1998, Frischer et al. (2000)measured mercury in marsh sediments adjacent to the site to aver-age 10 �g g−1. Therefore, mercury may still be present surroundingthe LCP Chemicals site and emitting gaseous mercury over a broadarea.

In July 2011, three 170 m transects were established in the saltmarsh west of and parallel to the LCP Chemicals site at distancesof 0.4, 0.6, and 1.3 km. Along each transect, Spanish moss trans-plants were tied to 15 PVC poles and placed 12.5 m apart. Eachtransplant consisted of four 5 g bundles of Spanish moss suspendedapproximately 2.5 m above sediment to avoid inundation by tides.At transplant placement, three air samples were collected (follow-ing the protocol described in Section 2.4.2) at the closest transect(0.4 km from the LCP Chemicals site) and three were collected at thefarthest transect (1.3 km from the site) at the center of each tran-sect. After two weeks, air sampling was repeated and transplantedSpanish moss samples were collected and fixed in nitric acid. Tis-sue mercury concentration of each sample was determined within48 h. Air mercury concentration was compared across transectsand times of transplant placement and collection using two-wayANOVA. Final tissue mercury concentration was compared to ini-tial concentrations and to the resident population at the site usingone-way ANOVA.

2.3.2. Transplants to coastal and inland sitesSpanish moss transplants were also placed near wetlands at

both coastal and inland sites in southeastern Georgia, USA to testwhether tissue mercury reflects air concentrations from nonpointsources over a wide geographic scale (approximately 14,000 km2;Fig. 1). At coastal sites (Sapelo Island, Ossabaw Island, and Cumber-land Island) transplants were placed within 100 m of salt marshes,while transplants at inland sites (Magnolia Springs and George L.Smith state parks, and a 45 ha, undeveloped tract of private forestedland) were placed near freshwater wetlands. Between June andSeptember 2011, 20 Spanish moss transplants (5 g) were tied toindividual trees 10 m apart along a 200 m transect at each site. Aftertwo weeks, 2.5 g of each transplant was collected (excluding Mag-nolia Springs state park and Ossabaw Island, which were collectedafter three and four weeks, respectively). In addition, Spanish mosssamples from the established, natural population were also col-lected for comparison to transplants at each site (with the exceptionof the private land that did not have a Spanish moss population).Spanish moss samples were placed immediately into nitric acid andanalyzed for mercury concentration within 24 h. Three air sampleswere collected in the center of each transect at times of trans-plant placement and collection except during transplant placementat Cumberland Island due to equipment malfunction. Final tissuemercury concentrations of transplants to coastal and inland siteswere compared to initial tissue levels and to resident populationspresent at the sites using either one-way ANOVA followed by TukeyHSD multiple comparisons or nonparametric Kruskal–Wallis testsfollowed by Mann–Whitney U-tests with a Bonferroni correctionfor multiple comparisons. Air mercury concentrations at transplantplacement and collection were compared using t-tests for eachcoastal and inland site.

2.3.3. Spanish moss and air sample collectionFor each study, 2.5 g Spanish moss samples were placed into

pre-weighed teflon vials with 6 ml nitric acid (HNO3) in the field to

fix mercury into solution, and transported on ice and in the dark sothat mercury concentrations would not change in transit to the lab-oratory (Southworth et al., 1958). Air samples were collected usinga diaphragm air pump to force 10 L of ambient air over 10 min into

K.T. Sutton et al. / Ecological Indicators 36 (2014) 392– 399 395

Table 1ANCOVA results for Spanish moss tissue Hg concentration of samples in retentionexperiment (Section 3.1).

df F p

Treatment 1 1229.89 <0.0001

au

2

2

w(dttat(m6s

2

gcV2fomo

3

3

ibmiwt

3

csttlwcfr

0

5

10

15

20

25

30

0 2 4 6 8 10 12 14 16

Tis

sue

Hg

Co

nce

ntr

ati

on

(µ

g g

-1)

Time (Days)

10x ambient

100x ambient

Fig. 2. Mean total mercury concentration in Spanish moss tissue over two weeks ina low atmospheric mercury environment. Tissue started with either 100× or 10×concentrations of mercury found in resident plants (n = 5).

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05

Tis

sue

Hg

Co

nce

ntr

ati

on

(µ

g g

)

Air Hg Concen tration (µg m )

Fig. 3. Relationship between mean air and tissue mercury concentrations in residentSpanish moss plants at urban (Jacksonville, �; Savannah, �), industrial (BrunswickLCP, ×), coastal (Ossabaw Island, �; Cumberland Island, ♦; Sapelo Island, ), andinland (George L. Smith state park, ©; and Magnolia Springs state park, �) sites.

Fig. 4. Mean mercury concentration of Spanish moss tissue prior to transplanting

4,56

Time 3 6.77 0.002Time × treatment 3 3.03 0.05

glass tube filled with gold sand to amalgamate mercury (Sutton,npublished data).

.4. Sample analysis

.4.1. TissueIn the laboratory, 0.5 ml of 201Hg stable isotope spike solution

as added to solutions of Spanish moss and HNO3 and microwavedCEM-MDS-2100) at 71.1 ◦C and a pressure of 30.4 kg cm−2 to breakown and liquefy tissue. An 0.5 ml aliquot of each sample washen passed through an Inductively-Coupled Plasma Mass Spec-rometer with a detection limit of 0.036 ng l−1, and the ratio of thedded 201Hg to the more abundantly occurring 202Hg stable iso-ope was compared to determine the total mercury concentration�g Hg g−1) in each tissue sample (Smith, 1993). NOAA CRM 2976

ussel tissue standards with a known mercury concentration of1.0 ± 0.0036 �g kg−1 were also used for comparison and protocoltandardization (Smith, 1993).

.4.2. AirA nichrome wire coil was placed around each gold sand-filled

lass tube and heated, which re-volatilized the amalgamated mer-ury. The re-volatilized mercury was swept by argon gas into a Coldapor Atomic Fluorescence Spectrophotometry detector (Tekran500), and an integrator (HP 3394) displayed peak fluorescencerom each air sample, with a detection limit of 0.13 ng l−1. Peak flu-rescence was compared to a standard curve of water with knownercury concentrations to calculate the air mercury concentration

f each sample (adapted from Bloom and Fitzgerald, 1988).

. Results

.1. Retention experiment

Following removal from a source of mercury vapor, Span-sh moss plants retained mercury for up to 15 days. Plants inoth the 100× and 10× treatments exhibited an increase inercury concentration over the depuration period (100×, mean

ncrease = 74.1 ± 17%; 10×, mean increase = 13.7 ± 11%), and thereas a strong trend toward a higher rate of increase in the100×

reatment compared to the 10× treatment (Table 1 and Fig. 2).

.2. Resident population study

There was a strong trend of increasing Spanish moss mercuryoncentrations with increasing air concentration across sites (Pear-on correlation; rp = 0.70; n = 8; p = 0.05; Fig. 3). The population fromhe industrial LCP Chemicals site had the highest mercury concen-ration both in air and in Spanish moss, while urban sites had theowest mercury levels. Air mercury concentrations within locations

ere highly variable relative to tissue mercury concentrations;

oefficients of variation (CV) for air were approximately twice thoseor Spanish moss tissue across sites (32.9–105.3% vs.16.9–60.6%,espectively).

(n = 10), after two weeks of exposure at three distances from the LCP Chemicals EPASuperfund site in Brunswick, GA (n = 15), and of a resident Spanish moss populationpresent at the site (n = 7). Error bars are ± one standard error of the mean (SEM).

3.3. Transplant studies

3.3.1. Transplants to a mercury-contaminated siteMercury concentrations in transplanted Spanish moss increased

by 164.8 ± 8.7% from initial concentrations after two weeks ofexposure to field conditions at the LCP site (one-way ANOVA;F = 20.7; p < 0.001; Fig. 4), and was similar to the resident pop-

ulation with a trend toward higher mercury concentration in theresident population (Tukey–Kramer HSD; 0.4 km distance, p = 0.14;0.6 km distance, p = 0.08; 1.3 km distance, p = 0.08). There was no

396 K.T. Sutton et al. / Ecological Indicators 36 (2014) 392– 399

A. Sapelo Isla nd

B. Cumberland Island

C. Ossabaw I sla nd

D. Private Land

E. Magnolia Springs

F. George L. Smith

0

0.05

0.1

0.15

0.2

0.25

0.3

C

B

A

0

0.05

0.1

0.15

0.2

0.25

0.3

A

A

B

0

0.05

0.1

0.15

0.2

0.25

0.3

A

A

A

0

0.05

0.1

0.15

0.2

0.25

0.3

B

A

0

0.05

0.1

0.15

0.2

0.25

0.3

BB

A

0

0.05

0.1

0.15

0.2

0.25

0.3

BB

A

Ini tial Ini tialFinal FinalResident Resident

Tis

sue

Hg C

on

cen

trati

on

(µ

g g

-1)

Tre atment Tre atment

F SEM in Spanish moss tissue transplanted to coastal (Sapelo Island [A], Cumberland Island[ ], and George L. Smith [F]) sites in comparison to the resident population concentration(

dcsawat

3

cFHtMvipmrpmHittpI

Table 2Total air mercury concentration (�g m−3) ± one standard error of the mean (SEM)measured during Spanish moss transplant placement and collection at each fieldsite (n = 4).

Site Placement CV% Collection CV%

Sapelo Island 0.007 ± 0.00 56.44 0.011 ± 0.00 44.55Cumberland Island – – 0.012 ± 0.00 52.69Ossabaw Island 0.004 ± 0.00 102.08 0.006 ± 0.00 89.83Private Land 0.075 ± 0.02 48.63 0.000 ± 0.00 2.00

ig. 5. Initial (n = 10) and final (n = 20) tissue mercury concentration (�g g−1) ± one

B], and Ossabaw Island [C]) and rural inland (Private Land [D], Magnolia Springs [En = 20) at each location.

ifference in air mercury concentration between placement andollection times (two-way ANOVA; F1,8 = 0.53; p = 0.49) or tran-ect location (two-way ANOVA; F1,8 = 1.16; p = 0.31). The averageir mercury concentration (combined initial and final air samples)as an order of magnitude higher than that for offshore ambient

ir (1.6 × 10−3 �g m−3), the standard for low air mercury concen-ration.

.3.2. Transplants to coastal and inland sitesThe mercury concentration of transplants increased from initial

oncentrations at inland sites Magnolia Springs (one-way ANOVA;2,46 = 44.22; p < 0.01) and George L. Smith (Kruskal–Wallis;2 = 17.44; p < 0.01), and reached final tissue mercury concen-

rations similar to the resident population (Tukey–Kramer HSD;S, p = 0.15; Mann–Whitney; GLS, p > 0.05; Fig. 5). At the pri-

ately owned inland site without a resident population, transplantsncreased from initial levels (t-test; t27 = −6.97, p < 0.01). Trans-lant responses at coastal sites differed by location (Fig. 5). Tissueercury concentrations increased in transplants to Sapelo Island

elative to initial concentrations (one-way ANOVA; F2,46 = 24.57; < 0.01; Tukey–Kramer HSD, p = 0.003) but did not reach tissueercury concentration of the resident population (Tukey–KramerSD; p < 0.001). Cumberland Island transplants remained sim-

lar to initial concentrations (Tukey–Kramer HSD, p = 0.92) but

ransplant mercury concentrations were only one-third that inissue from the resident population (Kruskal–Wallis; H2 = 19.17;

< 0.01; Mann–Whitney; p = 0.0006). Transplants to Ossabawsland exhibited no difference among initial and final tissue

Magnolia Springs 0.003 ± 0.00 32.86 0.041 ± 0.02 94.71George L. Smith 0.000 ± 0.00 0.00 0.006 ± 0.00 44.42

mercury concentrations of transplants and tissue concentration ofthe resident population (Kruskal–Wallis; H2 = 3.67; p = 0.16).

Air concentrations at inland sites were more variable andreached higher maximum values than at coastal sites (Table 2).Inland CV’s were 4-6X higher than coastal sites, and differed morebetween times of placement and collection than coastal sites. Forexample, the air concentration at Magnolia Springs was more vari-able during transplant collection than during placement (CV wasthree times higher), yet the private land site air concentration wasmore variable during placement (Table 2). The absolute air con-centrations at inland sites were higher during transplant collectionthan deployment at Magnolia Springs (t-test; t5 = 4.16; p = 0.009)and George L. Smith (t-test; t4 = −3.43; p = 0.03), but were lower at

the private inland site during transplant collection (t-test; t6 = 4.49;p = 0.004). In contrast, at Sapelo and Ossabaw Islands, the air con-centrations were similar between times of transplant placementand collection (t-test; t6 = −1.49; p = 0.19 and t-test; t6 = −0.63;

al Indi

pn(

4

esSicrchmcrmip

hrplocswcc(1ssm

crlLhlopt2amobpstepc

spdtts

K.T. Sutton et al. / Ecologic

= 0.55 respectively) and the direction of air concentration mag-itude increased from the time of sample placement and collectionTable 2).

. Discussion

We found that Spanish moss retains mercury in its tissue, anssential characteristic for a time-integrative bioindicator of atmo-pheric mercury concentrations. Other epiphytic plants, such asphagnum girgensohnii, also exhibit strong retention of mercuryn tissue for up to 4 weeks after removal from a source of mer-ury vapor (Lodenius et al., 2003). However, Spanish moss not onlyetained mercury regardless of air mercury concentration, but alsoontinued taking up mercury in the field over two weeks despiteaving reached high tissue concentrations. Therefore, saturation ofercury in tissue is unlikely to occur when exposed to ecologi-

ally relevant concentrations of atmospheric mercury. The similaretention and continued uptake of mercury over time by Spanishoss exposed to different concentrations should allow for time-

ntegrated measurements of atmospheric mercury, making thislant useful for development as a bioindicator.

As expected, areas with different land use and emission sourcesad differing atmospheric mercury concentrations, which wereeflected in resident Spanish moss tissue concentrations. The strongositive trend between air and tissue mercury concentrations is

ikely attributed to the long exposure time (and therefore periodf integration) of resident populations to atmospheric mercuryoncentrations. High variability in air concentration measured atites was expected due to the discrete sampling method used, inhich replicate one-time “snapshot” measurements of air mercury

oncentration were collected. Wind patterns, circulation, and pre-ipitation can impact the presence of gaseous mercury in an areaMorel et al., 1998; Rothenberg et al., 2010; Schroeder and Munthe,998). Therefore, air measurements taken at one point in time atites without a constant source of mercury are not likely to repre-ent average atmospheric mercury concentrations which may beeasurable using Spanish moss tissue concentrations.The positive trend between air and Spanish moss tissue mer-

ury concentration revealed the highest mercury concentrations inesident Spanish moss and in air at the Brunswick LCP site and theowest concentrations at the urban sites sampled. The BrunswickCP site was predicted to have elevated concentrations given itsistory of mercury contamination; however, our finding that urban

ocations had the least mercury in air and in resident Spanish mossf all sites sampled was unexpected. A possible explanation is thatrevailing wind patterns transported gaseous mercury to or fromarget sampling areas (Kellerhals et al., 2003; Rothenberg et al.,010). Prevailing winds in the area where samples were collectedre east- and northeastward (Weber and Blanton, 1980). Therefore,ercury emissions emanating from urban areas and sources west

r southwest of the urban centers may have been transported awayy winds at the time of sampling. For example, several coal-firedower plants are located within 20 km of the urban sites in ourtudy (US EPA, 2011). Plants such as these are estimated to con-ribute 22–33% of mercury in rainfall in the United States (Landingt al., 2010). Therefore, it is possible that prevailing winds trans-orted emissions from both the cities themselves, and from nearbyoal-fired power plants, offshore and away from urban areas.

Wind patterns alone cannot explain why rural inland and coastalites had higher mercury concentrations in air and in Spanish mossopulations than urban sites. Rural inland sites were not located

ownwind from urban sites. Soerensen et al. (2010) determinedhere is a 2–10× reduction in gaseous elemental mercury concen-ration in the atmosphere at distances of 40–120 km from emissionources, while Carballeira and Fernández (2002) found the lowest

cators 36 (2014) 392– 399 397

tissue mercury concentrations in the moss species (Scleropodiumpurum) 20–30 km from a coal-fired power plant. In the currentstudy, coal-fired power plants were ≥76 km from inland sites andtherefore unlikely to contribute to gaseous mercury concentra-tions measured at these sites. However, wetlands were presentthroughout the rural inland sites, and abundant bacteria in humicenvironments increase rates of mercury methylation (Benoit et al.,1999; Canário et al., 2007; King et al., 2001). Methylated speciescan then be taken up by organisms, or de-methylated to Hg (II) andHg0 and re-volatilized (Langer et al., 2001; Lindberg et al., 2002).Therefore, local atmospheric mercury concentration is likely to beelevated around wetlands (Williams et al., 1994; Zillioux et al.,1993).

Tissue mercury content of Spanish moss transplanted to siteswith and without known mercury emission sources reflected airmercury concentrations. At the Brunswick LCP site, mean airmercury concentration in the marsh surrounding the site was con-sistently an order of magnitude greater than background levels.In addition, tissue concentrations in both transplants and the res-ident population of Spanish moss at the LCP Chemicals site wereelevated 3–4X above background levels reported in the literaturefor Spanish moss (0.1 �g g−1; Calasans and Malm, 1997) and forthe moss species Pleurozium schreberi (0.06–0.09 �g g−1; Rinne andBarclay-Estrup, 1980). Further, there was no difference in mercuryconcentration in Spanish moss transplant or air measurements upto 1.3 km from the site. These results suggest mercury emissionfrom the contaminated sediments throughout the site, particularlysince Malm et al. (1998) found mercury concentrations in Spanishmoss plants decreased 33-fold within 20 m from gold shops (pointsources of mercury emissions). It is possible that an atmosphericevent brought elevated concentrations of atmospheric mercury tothe site during the experiment. However, the trend toward highertissue mercury concentration in the resident population comparedto transplanted plants provides evidence that the area likely servesas a constant source of gaseous mercury emissions, and indicatedthat this plant is capable of rapidly responding to a constant, butdiffuse source of mercury in the field.

At coastal and inland sites Spanish moss transplants were sen-sitive to even low levels of mercury from environments without aconstant source of mercury emission. With the exception of Cum-berland and Ossabaw Islands, transplanted tissue had increasedmercury concentrations relative to starting concentrations. Thecoastal site most likely influenced by emission sources transportedby August northeastward winds was Sapelo Island. This site islocated 30.5 km northeast of a coal fired power plant, and 35.5 kmnortheast of the Brunswick LCP Chemicals site (US EPA, 2011).However, transplants to inland sites with less potential to receivewind-borne mercury also showed increased tissue mercury con-centrations. Coastal sites did not have higher concentrations ofmercury in air and in Spanish moss than inland sites despite thepresence of greater amounts of wetland habitat at all sites. Morevariable wind patterns on the coast (Weber and Blanton, 1980) mayhave resulted in lower short-term coastal atmospheric mercuryconcentrations, reflected in lower tissue concentrations of trans-plants.

5. Conclusions

In this study we found Spanish moss has characteristics usefulfor development as a bioindicator of atmospheric mercury concen-trations. Spanish moss retains mercury in its tissue regardless of

air concentration, and the positive trend between mercury con-centration in resident Spanish moss tissue and in air indicates thatthis plant can be used to detect patterns in atmospheric concen-tration over both long periods of time and on a wide geographic

3 al Indi

ssmdetswciraIlsbcmcaHast

A

afWasAi

A

fjm

R

A

A

B

B

B

B

B

C

C

98 K.T. Sutton et al. / Ecologic

cale. Transplants integrate atmospheric concentrations into tis-ue, and could potentially be used to assess average atmosphericercury concentrations in locations where there is no historical

ata. Further, our transplant studies suggest Spanish moss may beffective in monitoring changes in atmospheric mercury concen-rations from diffuse sources on the order of weeks, to monitorhorter-term changes in atmospheric concentrations (e.g. duringildfire events, or seasonal changes). The increased mercury con-

entration in Spanish moss at the polluted Brunswick LCP sitendicated that Spanish moss responds rapidly to changes in envi-onmental mercury concentrations due to constant emissions from

nonpoint source while transplants at inland sites and at Sapelosland revealed that Spanish moss takes up mercury present inow, ambient concentrations at areas not adjacent to a knownource. Therefore, our findings suggest that Spanish moss cane used to compare ecologically relevant atmospheric mercuryoncentrations across sites. In order to use Spanish moss tissueercury concentration as a quantitative measure of atmospheric

oncentration, the direct relationship between air concentrationnd mercury uptake by Spanish moss needs to be established.owever, a qualitative Spanish moss bioindicator that integratestmospheric mercury concentrations from nonpoint sources bothpatially and temporally can provide a valuable and cost-effectiveool to supplement existing monitoring programs.

cknowledgments

We would like to thank Georgia Power (GPC Agreement #16658)nd the Georgia Southern University College of Graduate Studiesor providing funding for this study. We would also like to thank H.

indom, C.R. Chandler, and C. Alexander for their valuable inputnd assistance. Special thanks to D. Wells, C. Robertson, J. John-on, C. Little and J. Cain for technical expertise. Finally, we thank R.chtziger and two anonymous reviewers for comments that greatly

mproved the manuscript.

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.ecolind.2013.08.011. These data include Google maps of the

ost important areas described in this article.

eferences

lves, E.S., Moura, B.B., Domingos, M., 2008. Structural analysis of Tillandsia usneoidesL. exposed to air pollutants in São Paulo City-Brazil. Water Air Soil Pollut. 189,61–68.

mado Filho, G.M., Andrade, L.R., Farina, M., Malm, O., 2002. Hg localization inTillandsia usneoides L. (Bromeliaceae) an atmospheric biomonitor. Atmos. Envi-ron. 36, 881–887.

aker, A.J.M., 1981. Accumulators and excluders – strategies in the response of plantsto heavy metals. J. Plant Nutr. 3, 643–654.

astos, W.R., Fonseca, M.F., Pinto, F.N., Rebelo, M.F., dos Santos, S.S., da Silveira,E.G., Torres, J.P., Malm, O., Pfeiffer, W.C., 2004. Mercury persistence in indoorenvironments in the Amazon Region, Brazil. Environ. Res. 96, 235–238.

enoit, J.M., Gilmour, C.C., Mason, R.P., Heyes, A., 1999. Sulfide controls on mercuryspeciation and bioavailability to methylating bacteria in sediment pore waters.Environ. Sci. Technol. 33, 951–957.

loom, N., Fitzgerald, W.F., 1988. Determination of volatile mercury species atthe picogram level by low-temperature gas chromatography with cold-vaporatomic fluorescence detection. Anal. Chim. Acta 208, 151–161.

righigna, L., Ravanelli, M., Minelli, A., Ercoli, L., 1997. The use of an epiphyte (Tilland-sia caput-medusae morren) as bioindicator of air pollution in Costa Rica. Sci. TotalEnviron. 198, 175–180.

alasans, C.F., Malm, O., 1997. Elemental mercury contamination survey in a chlor-alkali plant by the use of transplanted Spanish moss Tillandsia usneoides (L.). Sci.Total Environ. 208, 165–177.

anário, J., Vale, M.C.C., Cesário, R., 2007. Evidence for elevated production ofmethylmercury in salt marshes. Environ. Sci. Technol. 41, 7376–7382.

cators 36 (2014) 392– 399

Carballeira, A., Fernández, J.A., 2002. Bioconcentration of metals in the moss Scle-ropodium purum in the area surrounding a power plant: a geotopographicalpredictive model for mercury. Chemosphere 47, 1041–1048.

Conti, M.E., Cecchetti, G., 2001. Biological monitoring: lichens as bioindicators of airpollution assessment—a review. Environ. Pollut. 114, 471–492.

Davis, D.D., McClenahen, J.R., Hutnik, R.J., 2007. Use of the moss Dicranum montanumto evaluate recent temporal trends of mercury accumulation in oak forests ofPennsylvania. Northeast. Nat. 14, 27–34.

De Temmerman, L., Bell, J.N.B., Garrec, J.P., Klumpp, A., Krause, G.H.M., Tonneijck,A.E.G., 2004. Biomonitoring of air pollution with plants – considerations forthe future. In: Klumpp, A., Ansel, W., Klumpp, G. (Eds.), Urban Air Pollution,Bioindication, and Environmental Awareness. Cuvillier Verlag, Göttingen, pp.337–373.

Falla, J., Laval-Gilly, P., Henryon, M., Morlot, D., Ferard, J., 2000. Biological air qualitymonitoring: a review. Environ. Monit. Assess. 64, 627–644.

Figueiredo, A.M.G., Alcalá, A.L., Ticianelli, R.B., Domingos, M., Saiki, M., 2004. The useof Tillandsia usneoides L. as bioindicator of air pollution in São Paulo, Brazil. J.Radioanal. Nucl. Chem. 259, 59–63.

Figueiredo, A.M.G., Nogueira, C.A., Saiki, M., Milian, F.M., Domingos, M., 2007. Assess-ment of atmospheric metallic pollution in the metropolitan region of São Paulo,Brazil, employing Tillandsia usneoides L. as biomonitor. Environ. Pollut. 145,279–292.

Frischer, M.E., Danforth, J.M., Healy, M.A.N., Saunders, F.M., 2000. Whole-cell versustotal RNA extraction for analysis of microbial community structure with 16SrRNA-targeted oligonucleotide probes in salt marsh sediments. Appl. Environ.Microbiol. 66, 3037–3043.

Gardner, W.S., Kendall, D.R., Odom, R.R., Windom, H.L., Stephens, J.A., 1978. The dis-tribution of methyl mercury in a contaminated salt marsh ecosystem. Environ.Pollut. 15, 243–251.

Grangeon, S., Guédron, S., Asta, J., Sarret, G., Charlet, L., 2012. Lichen and soil asindicators of an atmospheric mercury contamination in the vicinity of a chlor-alkali plant (Grenoble, France). Ecol. Indic. 20, 163–169.

Grönlund, R., Sjöholm, M., Weibring, P., Edner, H., Svanberg, S., 2005. Elemental mer-cury emissions from chlor-alkali plants measured by lidar techniques. Atmos.Environ. 39, 7474–7480.

Harmens, H., Norris, D.A., Steinnes, E., Kubin, E., Piispanen, J., Alber, R., Aleksiayenak,Y., Blum, O., Cos kun, M., Dam, M., De Temmerman, L., Fernández, J.A., Frolova,M., Frontasyeva, M., González-Miqueo, L., Grodzinska, K., Jeran, Z., Korzekwa,S., Krmar, M., Kvietkus, K., Leblond, S., Liiv, S., Magnússon, S.H., Mankovaská, B.,Pesch, R., Rühling, Å., Santamaria, J.M., Schröder, W., Spiric, Z., Suchara, I., Thöni,L., Urumov, V., Yurukova, L., Zechmeister, H.G., 2010. Mosses as biomonitors ofatmospheric heavy metal deposition: spatial patterns and temporal trends inEurope. Environ. Pollut. 158, 3144–3156.

Husk, G.J., Weishampel, J.F., Schlesinger, W.H., 2004. Mineral dynamics in Spanishmoss, Tillandsia usneoides L. (Bromeliaceae), from Central Florida, USA. Sci. TotalEnviron. 321, 165–172.

Kellerhals, M., Beauchamp, S., Belzer, W., Blanchard, P., Froude, F., Harvey, B., McDon-ald, K., Pilote, M., Poissant, L., Puckett, K., Schroeder, B., Steffen, A., Tordon,R., 2003. Temporal and spatial variability of total gaseous mercury in Canada:results from the Canadian Atmospheric Mercury Measurement Network (CAM-Net). Atmos. Environ. 37, 1003–1011.

King, J.K., Kostka, J.E., Frischer, M.E., Saunders, F.M., Jahnke, R.A., 2001. A quantitativerelationship that demonstrates mercury methylation rates in marine sedimentsare based on the community composition and activity of sulfate-reducing bacte-ria. Environ. Sci. Technol. 35, 2491–2496.

Kłos, A., Rrajfur, M., Srámek, I., Wacławek, M., 2012. Mercury concentration inlichen, moss and soil samples collected from the forest areas of Praded andGlacensis Euroregions (Poland and Czech Republic). Environ. Monit. Assess. 184,6765–6774.

Landing, W.M., Caffrey, J.M., Nolek, S.D., Gosnell, K.J., Parker, W.C., 2010. Atmosphericwet deposition of mercury and other trace elements in Pensacola, Florida. Atmos.Chem. Phys. 10, 4867–4877.

Langer, C.S., Fitzgerald, W.F., Visscher, P.T., Vandal, G.M., 2001. Biogeochemicalcycling of methylmercury at Barn Island Salt Marsh, Stonington, CT, USA. Wetl.Ecol. Manage. 9, 295–310.

Lindberg, S.E., Dong, W., Meyers, T., 2002. Transpiration of gaseous elemental mer-cury through vegetation in a subtropical wetland in Florida. Atmos. Environ. 36,5207–5219.

Lodenius, M., Tulisalo, E., Soltanpour-Gargari, A., 2003. Exchange of mercurybetween atmosphere and vegetation under contaminated conditions. Sci. TotalEnviron. 304, 169–174.

Malm, O., Fonseca, M.F., Miguel, P.H., Bastos, W.R., Pinto, F.N., 1998. Use of epiphyteplants as biomonitors to map atmospheric mercury in a gold trade center city,Amazon, Brazil. Sci. Total Environ. 213, 57–64.

Martin, C.E., Christensen, N.L., Strain, B.R., 1981. Seasonal patterns of growth,tissue acid fluctuations, and 14CO2 uptake in the crassulacean acidmetabolism epiphyte Tillandsia usneoides L. (Spanish moss). Oecologia 49,322–328.

Morel, F.M.M., Kraepiel, A.M.L., Amyot, M., 1998. The chemical cycle and bioaccu-mulation of mercury. Annu. Rev. Ecol. Syst. 29, 543–566.

O’Driscoll, N.J., Poissant, L., Canário, J., Ridal, J., Lean, D.R.S., 2007. Continuous anal-

ysis of dissolved gaseous mercury and mercury volatilization in the upper St.Lawrence River: exploring temporal relationships and UV attenuation. Environ.Sci. Technol. 41, 5342–5348.

Rinne, R.J.K., Barclay-Estrup, P., 1980. Heavy metals in feather moss, Pleuroziumschreberi, and in soils in NW Ontario, Canada. OIKOS 34, 59–67.

al Indi

R

S

S

S

S

S

S

U

K.T. Sutton et al. / Ecologic

othenberg, S.E., McKee, L., Gilbreath, A., Yee, D., Connor, M., Fu, X., 2010. Evidencefor short-range transport of atmospheric mercury to a rural, inland site. Atmos.Environ. 44, 1263–1273.

chroeder, W.H., Munthe, J., 1998. Atmospheric mercury—an overview. Atmos. Envi-ron. 32, 809–822.

mith, R.G., 1993. Determination of mercury in environmental samples by isotopedilution/ICPMS. Anal. Chem. 65, 2485–2488.

oerensen, A.L., Skov, H., Johnson, M.S., Glasius, M., 2010. Gaseous mercury in coastalurban areas. Environ. Chem. 7, 537–547.

outhworth, B.C., Hodecker, J.H., Fleisher, K.D., 1958. Determination of mer-cury in organic compounds: a micro and semimicromethod. Anal. Chem. 30,1152–1153.

teinnes, E., Berg, T., Sjøbakk, T.E., 2003. Temporal and spatial trends inHg deposition monitored by moss analysis. Sci. Total Environ. 304,

215–219.

uchara, I., Sucharová, J., 2008. Mercury distribution around the Spolana chlor-alkaliplant (central Bohemia, Czech Republic) after a catastrophic flood, as revealedby bioindicators. Environ. Pollut. 151, 352–361.

nited States Census Bureau (US Census), 2011. http://quickfacts.census.gov/qfd/

cators 36 (2014) 392– 399 399

United States Environmental Protection Agency (US EPA), 2011.http://www.epa.gov/superfund/sites/npl/nar1458.htm

Wagemann, R., Lockhart, W.L., Welch, H., Innes, S., 1995. Arctic marine mammalsas integrators and indicators of mercury in the arctic. Water Air Soil Pollut. 80,683–693.

Wannaz, E.D., Carreras, H.A., Pérez, C.A., Pignata, M.L., 2006. Assessment of heavymetal accumulation in two species of Tillandsia in relation to atmospheric emis-sion sources in Argentina. Sci. Total Environ. 361, 267–278.

Weber, A.H., Blanton, J.O., 1980. Monthly mean wind fields for the South AtlanticBight. J. Phys. Oceanogr. 10, 1256–1263.

Williams, T.P., Bubb, J.M., Lester, J.N., 1994. Metal accumulation within salt marshenvironments: a review. Mar. Pollut. Bull. 28, 277–290.

Windom, H., Gardner, W., Stephens, J., Taylor, F., 1976. The role of methylmercuryproduction in the transfer of mercury in a salt marsh ecosystem. Estuar. Coast.

Mar. Sci. 4, 579–583.

Winger, P.V., Lasier, P.J., Geitner, H., 1993. Toxicity of sediments and pore water fromBrunswick Estuary, Georgia. Arch. Environ. Contam. Toxicol. 25, 371–376.

Zillioux, E.J., Porcella, D.B., Benoit, J.M., 1993. Mercury cycling and effects in fresh-water wetland ecosystems. Environ. Toxicol. Chem. 12, 2245–2264.