Ecotoxicology and Environmental Safety 97 (2013) 154–159

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Ecotoxicology and Environmental Safety

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n CorrE-m

journal homepage: www.elsevier.com/locate/ecoenv

Effect of metal stress on photosynthetic pigments in theCu-hyperaccumulating lichens Cladonia humilis andStereocaulon japonicum growing in Cu-polluted sites in Japan

Hiromitsu Nakajima a,n, Yoshikazu Yamamoto b, Azusa Yoshitani b, Kiminori Itoh a

a Graduate School of Environment and Information Sciences, Yokohama National University, Tokiwadai 79-7, Hodogayaku, 240-8501 Yokohama, Japanb Graduate School of Bioresource Sciences, Akita Prefectural University, Shimoshinjyo-nakano, 010-0195 Akita, Japan

a r t i c l e i n f o

Article history:Received 27 March 2013Received in revised form20 July 2013Accepted 24 July 2013Available online 15 August 2013

Keywords:Cu pollutionAl stressICP-MSUV–vis absorption spectraPigmentsSecondary metabolites

13/$ - see front matter & 2013 Elsevier Inc. Alx.doi.org/10.1016/j.ecoenv.2013.07.026

esponding author. Fax: +81 45 339 4354.ail address: h-nakaji@ynu.ac.jp (H. Nakajima).

a b s t r a c t

To understand the ecology and physiology of metal-accumulating lichens growing in Cu-polluted sites,we investigated lichens near temple and shrine buildings with Cu roofs in Japan and found thatStereocaulon japonicum Th. Fr. and Cladonia humilis (With.) J. R. Laundon grow in Cu-polluted sites. Metalconcentrations in the lichen samples collected at some of these sites were determined by inductivelycoupled plasma mass spectroscopy (ICP-MS). UV–vis absorption spectra of pigments extracted from thelichen samples were measured, and the pigment concentrations were estimated from the spectral datausing equations from the literature. Secondary metabolites extracted from the lichen samples wereanalyzed by high-performance liquid chromatography (HPLC) with a photodiode array detector. Wefound that S. japonicum and C. humilis are Cu-hyperaccumulating lichens. Differences in pigmentconcentrations and their absorption spectra were observed between the Cu-polluted and controlsamples of the 2 lichens. However, no correlation was found between Cu and pigment concentrations.We observed a positive correlation between Al and Fe concentrations and unexpectedly found highnegative correlations between Al and pigment concentrations. This suggests that Al stress reducespigment concentrations. The concentrations of secondary metabolites in C. humilis growing in the Cu-polluted sites agreed with those in C. humilis growing in the control sites. This indicates that themetabolite concentrations are independent of Cu stress.

& 2013 Elsevier Inc. All rights reserved.

1. Introduction

Lichens are valuable for monitoring and assessing metal pollutionbecause many lichen species grow in metal-polluted environmentsand accumulate metals (Purvis and James, 1985; Nash, 1990; Purvisand Halls, 1996, Pawlik-Skowronska et al., 2008; Purvis, 2010;Rajakaruna et al., 2011). Hence, understanding the ecology andphysiology of metal-accumulating lichens is important for monitoringand assessing metal pollution. Cu is one of the most toxic metals tofungi and algae (Nieboer and Richardson, 1980). However, 97 speciesof lichen were found in Cu-polluted sites around an abandoned Cumine in England (Purvis and James, 1985). The genera Cladonia andStereocaulon are common in metal-polluted sites (Nash, 1990); 12Cladonia species are tolerant to Cu and Zn (Tyler, 1989) and 4Stereocaulon species occur on metal-rich rock (Dobson, 2005). How-ever, information regarding the lichen flora in Cu-polluted areas ofJapan is still lacking, and little is known about the Cladonia andStereocaulon genera in such areas. In a previous study, we investigated

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Cu-polluted sites in Japan and found that the cup lichens Cladoniahumilis (With.) J. R. Laundon and Cladonia subconistea Asahina grow onthe Cu-hyperaccumulating moss Scopelophila cataractae (Mitt.) Broth.(Nakajima et al., 2012). This is the first report on the 2 lichens growingin Cu-polluted sites, which was unexpected for C. humilis, because it iswidely distributed throughout Africa, Asia, Australasia, Europe as wellas North and South America and has been observed in normalenvironments thus far (Nash et al., 2002). On the other hand,C. subconistea occurs only in Asia (Yoshimura, 1974; Abbas et al.,2001; Nayaka et al., 2009; Baniya et al., 2010; Wang et al., 2011). In theprevious study, we discussed the qualitative effects of Cu stress onphotosynthetic pigments in the 2 lichens; however, we did notexamine the correlations between Cu and pigment concentrations(Nakajima et al., 2012). Moreover, we did not consider the effects ofother metals and effects of Cu and other metals on lichen secondarymetabolites. Therefore, further investigation of Cu-polluted sites inJapan should provide new valuable information regarding the lichenflora at these sites, allow the quantitative analysis of metals andsecondary metabolites in the lichens, and provide a deeper under-standing of the ecology and physiology of lichens.

The purpose of the present study was to identify lichensgrowing at Cu-polluted sites in Japan and determine the effects

H. Nakajima et al. / Ecotoxicology and Environmental Safety 97 (2013) 154–159 155

of Cu and other metals on their photosynthetic pigments andsecondary metabolites. Hence, we investigated Cu-polluted sites inJapan more thoroughly than the previous study and found that thefruticose lichens Stereocaulon japonicum Th. Fr. and C. humilis growin Cu-polluted sites other than those examined in the previousstudy. This was the first observation of S. japonicum growing at aCu-polluted site. We determined the concentrations of 8 metals(Cu, Na, Mg, Ca, Mn, Fe, and Zn, except Al, are essential in lichens(Ahmadjian, 1993)) in the lichen samples collected at some ofthese sites, analyzed pigments and secondary metabolitesextracted from the lichen samples, and examined the effects ofmetals on these pigments and metabolites.

Metal-induced stress in lichens can be assessed by analyzingtheir pigments (Garty, 2001; Boonpragob, 2002; Bačkor andFahselt, 2004; Bačkor et al., 2009). Specifically, pigment absor-bance ratios have been used to assess metal stress in lichens. Oneof these ratios is the chlorophyll a/b ratio, which is decreased byCu stress (Chettri et al., 1998) and another is the ratio ofabsorbance at 435 and 415 nm, which represents the degradationof chlorophyll a to pheophytin a (Garty, 2001). These ratios can bedetermined by measuring the absorption spectra of pigmentsextracted from lichens (Ronen and Galun, 1984; Wellburn, 1994).However, the absorption spectra themselves have not been fullyexamined. In the previous study, we observed significant differ-ences in the absorption spectra of control and Cu-contaminatedlichen samples. This observation suggested that Cu contaminationof a lichen sample could be determined from its absorption spectra(Nakajima et al., 2012).

Over 700 lichen secondary metabolites have been identified,and this diversity provides a useful chemical fingerprint to helpidentify lichens (Purvis, 2000). Furthermore, lichen secondarymetabolites are sensitive to heavy metal accumulation, and theirsensitivity is species-specific (Molnár and Farkas, 2010). A correla-tion has been reported between the concentrations of secondarymetabolites and metals (Pawlik-Skowronska and Backor, 2011).The accumulation of Cu in S. japonicum and C. humilis may beassociated with the presence of particular secondary metabolites:atranorin, stictic acid, and norstictic acid in S. japonicum andatranorin and fumarprotocetraric acid in C. humilis (Yoshimura,1974).

2. Materials and methods

2.1. Sampling and sample identification

Lichen samples were collected in March–September 2012 from 7 Cu-pollutedsites in Isehara, Takao, Ueno, and Tsukuba and 6 control sites in Isehara, Katsushika,and Chofu in Japan. At most sampling sites, the lichen colony sizes wereapproximately 10 cm or less in diameter; consequently, the sample amountscollected at some of the sites were not large enough to perform all of thesubsequent analyses. For example, the lichen samples collected at some of thesites were analyzed by UV–vis spectroscopy but not by inductively coupled plasmamass spectroscopy (ICP-MS). All the samples were dried in paper envelopes for2 weeks under normal laboratory conditions. The samples were identified by theirmorphology and secondary metabolites; the analysis of the latter is described inthe next section.

2.2. Analysis of lichen secondary metabolites

Secondary metabolites were extracted from dried samples overnight in acetone.The sample concentration was 5.0 mg/ml. The acetone solution was analyzed byhigh-performance liquid chromatography (HPLC) with a photodiode array detector.HPLC conditions were as follows: column, YMC-Pack ODS-A (150 mm�4.6 mm i.d.,5 μm, 12 nm) at 40 1C; solvent, MeOH:H2O:H3PO4¼80:20:1 at 1 ml/min; detectionwavelength, 254 nm; and wavelength range of the UV spectrum, 200–400 nm. Peakswere detected at retention times corresponding to atranorin, stictic acid, andnorstictic acid for S. japonicum and atranorin and fumarprotocetraric acid for C.humilis. The relative concentrations of these secondary metabolites were representedby peak areas at the specific retention times corresponding to each metabolite.

2.3. Analysis of metals

The concentrations of 8 metals, Cu, Na, Mg, Al, Ca, Mn, Fe, and Zn, in pseudopodetiaof S. japonicum and podetia of C. humilis were determined by ICP-MS. Pseudopodetiaand podetia were dried at 90 1C for 24 h. Approximately 10 and 40mg of the driedsamples collected at the Cu-polluted and control sites, respectively, were digested in65% HNO3 and 30% H2O2 (2:1, v/v) for 24 h, and the volumes were made up to 20 mlwith deionized water. Metal concentrations were measured using an ICP-MS instru-ment (Agilent 7700x).

2.4. Analysis of UV–vis absorption spectra of pigments

Pigments were extracted from 5 mg of dried samples in 2 ml of dimethylsulfoxide (DMSO) for 60 min at 65 1C in the dark. After cooling to room tempera-ture, the absorption spectra of the pigments in DMSO were measured using aspectrophotometer. Chlorophyll concentrations were calculated using Wellburn'sequations (Wellburn, 1994) and the absorbance values at 480, 649, and 665 nm.The degradation of chlorophyll a to pheophytin awas evaluated by determining theratio of the absorbance at 435 and 415 nm (A435/A415) (Garty, 2001).

2.5. Statistical analysis

Correlations between two parameters were tested by linear regression analysisusing Microsoft Excel. Significant differences were determined by t-test using thesoftware.

3. Results

3.1. Lichen species in Cu-polluted sites

We investigated lichen species at Cu-polluted sites in Japanaround Cu-roofed temples and shrines and found a fruticoselichen, S. japonicum, growing on a stone wall in Isehara (KanagawaPrefecture). The Isehara site is located approximately 500 mabove sea level halfway up a mountain. At the site, S. japonicumgrew on a stone wall under the Cu-roofed temple building,the colony of which was adjacent to a moss mat of S. cataractae(a Cu-hyperaccumulator) on the stone wall.

We also observed C. humilis growing on soil in Isehara (Kana-gawa Prefecture), Takao (Tokyo), Tsukuba (Ibaraki Prefecture), andUeno (Tokyo) and on soil and moss in Nagoya (Aichi Prefecture).This Isehara site is near the Isehara site mentioned above. At thisIsehara site, C. humilis grew on soil near a moss mat of S. cataractaeunder a Cu-roofed temple building. The Takao and Tsukuba sitesare located approximately 500 and 300 m above sea level, respec-tively, halfway up mountains, whereas the Ueno and Nagoya sitesare located at near sea level in urban areas. At these 4 sites,C. humilis grew on soil (at the Nagoya site alone, C. humilis grew onsoil and on a moss mat of S. cataractae) near Cu-roofed templebuildings, under which S. cataractae occurred.

3.2. Metal analysis

The metal concentrations in lichen samples are listed in Table 1.We found that S. japonicum growing in the Isehara site is highly

contaminated with Cu. The Cu concentration of S. japonicumgrowing on a stone wall under the Cu-roofed temple building atIsehara was 1.7�102 times higher than that of S. japonicumgrowing on rock in the control site at Katsushika. No such largedifferences in the concentrations of any of other metals wereobserved between the Cu-polluted and the control samples.

On the other hand, we found that C. humilis growing in theTakao and Nagoya sites is heavily contaminated with Cu. Theaverage Cu concentration of C. humilis growing in the Cu-pollutedsites was 63 times higher than that of C. humilis growing in thecontrol sites. Similar to what was observed for S. japonicum, therewere no such large differences in the concentrations of other

Table 1Metal concentrations of S. japonicum and C. humilis growing in the control and Cu-polluted sites. Values are average (standard deviation) (n¼3).

Species Site Substrate Metal concentration [mg/kg dry weight]

Na Mg Al Ca Mn Fe Cu Zn

S. japonicum Control Katsushika-1 Rock 237.2 (5.5) 219.3 (3.4) 726.9 (7.8) 155.5 (2.8) 12.8 (0.1) 602.0 (2.0) 7.6 (0.1) 77.1 (0.3)S. japonicum Cu-polluted Isehara-1 Stonewall 195.3 (2.5) 357.3 (2.2) 284.5 (3.2) 839.8 (10.3) 7.1 (2.2) 257.3 (2.7) 1296.6 (13.3) 70.5 (0.9)C. humilis Control Isehara-2 Soil 310.5 (5.7) 772.7 (6.1) 145.8 (4.1) 2092.5 (8.0) 6.8 (7.2) 119.5 (2.8) 8.4 (3.1) 451.8 (3.4)C. humilis Control Katsushika-2 Rock 313.3 (0.7) 846.9 (1.5) 642.0 (3.1) 2907.7 (3.2) 42.6 (0.5) 743.7 (3.7) 5.4 (0.1) 76.5 (0.7)C. humilis Control Katsushika-3 Rock 275.5 (5.9) 704.7 (16.7) 354.6 (10.7) 2258.5 (57.0) 84.4 (1.8) 374.1 (11.0) 2.0 (2.5) 98.1 (2.0)C. humilis Cu-polluted Takao Soil 256.4 (1.1) 701.8 (4.5) 1116.1 (7.9) 2237.6 (27.0) 80.4 (6.5) 1046.9 (8.2) 74.9 (3.0) 366.1 (3.7)C. humilis Cu-polluted Nagoya-1 Moss 454.1 (7.8) 809.5 (5.6) 536.5 (10.4) 4789.1 (67.0) 55.2 (1.0) 612.0 (15.0) 612.7 (11.7) 261.7 (3.5)C. humilis Cu-polluted Nagoya-2 Soil 230.1 (2.7) 956.3 (13.8) 276.9 (3.2) 4516.8 (67.2) 78.4 (1.2) 171.6 (2.6) 311.6 (2.7) 241.8 (1.9)

Fig. 1. Representative absorption spectra of pigments extracted from the controland Cu-polluted samples of S. japonicum.

Fig. 2. Representative absorption spectra of pigments extracted from Cu-polluted(a) and control (b) samples of C. humilis.

H. Nakajima et al. / Ecotoxicology and Environmental Safety 97 (2013) 154–159156

metals between the Cu-polluted and the control samples of C.humilis.

To understand the relationships between metal concentrationsin the lichen samples, correlation coefficients (R) and p-values (p)were calculated between the metal concentrations in C. humilis.The highest correlation was observed between Al and Fe (R¼0.974,po0.01) followed by Ca and Cu (R¼0.910, po0.05).

3.3. UV–vis absorption spectra of pigments

Fig. 1 shows representative UV–vis absorption spectra of pig-ments extracted from S. japonicum growing in the Cu-polluted andcontrol sites. The absorption spectra of the Cu-polluted samplesagreed with those of the control samples, except for the differ-ences observed at shorter wavelengths.

In contrast, the absorption spectra of pigments extracted fromC. humilis growing in the Cu-polluted sites differed significantlyfrom those extracted from C. humilis growing in the control sites;for the Cu-polluted samples, the absorbance of the band at 387 nmis greater than that at 407 nm (Fig. 2a), whereas for the controlsamples, the absorbance of the band at 387 nm is slightly greateror less than that at 407 nm (Fig. 2b). This difference in absorbancesat 387 and 407 nm for the Cu-polluted samples (0.04870.014 for12 samples at 6 Cu-polluted sites) is 3 times greater (po0.01) thanthat for the control samples (0.01570.020 for 12 samples at4 control sites).

3.4. Pigment analysis

The concentrations of chlorophyll a and b, the chlorophyll a/bratio, the concentration of total carotenoids, and the A435/A415 ratio

were obtained using the spectral data, and the results aresummarized in Table 2.

The chlorophyll a/b ratio of S. japonicum growing in the Cu-polluted site was 18% less than that of S. japonicum growing in thecontrol sites (2.8470.09 for 6 samples at 2 control sites). Theconcentrations of chlorophyll a and b in the Cu-polluted sampleswere 1.3 and 1.6 times higher, respectively, than those in the controlsamples. However, the concentration of total carotenoids and theA435/A415 ratio of the Cu-polluted samples agreed with those of thecontrol samples (0.5570.10 and 1.2170.08, respectively).

On the contrary, the chlorophyll a/b ratio and the concentrationsof chlorophyll a and b in C. humilis growing in the Cu-pollutedsites (2.9370.13, 1.3770.49, and 0.4870.18, respectively, for 12samples at 6 Cu-polluted sites) agreed with those growing in the

Table 2Concentrations of chlorophyll (chl.) a and b in dry weight (DW), chl. a/b ratio, concentration of total carotenoids (X+C) in DW, and the ratio of absorbance at 435 and 415 nm(A435/A415). Values are average (standard deviation) (n¼3) or single value (n¼1).

Species Site Chl. a [mg/g] Chl. b [mg/g] Chl. a/b X+C [mg/g] A435/A415

S. japonicum Control Katsushika-1 1.54 (0.09) 1.54 (0.09) 2.79 (0.02) 0.62 (0.04) 1.28 (0.02)S. japonicum Control Tsukuba-1 1.09 (0.33) 0.55 (0.04) 2.88 (0.12) 0.47 (0.10) 1.15 (0.06)S. japonicum Cu-polluted Isehara-1 1.69 (0.32) 0.72 (0.09) 2.34 (0.15) 0.55 (0.09) 1.17 (0.06)C. humilis Control Isehara-2 2.36 (0.44) 0.70 (0.14) 3.41 (0.07) 0.73 (0.12) 0.96 (0.02)C. humilis Control Katsushika-2 1.43 (0.33) 0.49 (0.10) 2.89 (0.06) 0.51 (0.11) 1.17 (0.03)C. humilis Control Katsushika-3 1.48 (0.07) 0.53 (0.01) 2.78 (0.10) 0.52 (0.04) 1.01 (0.01)C. humilis Control Chofu 1.47 (0.19) 0.49 (0.06) 2.99 (0.04) 0.55 (0.07) 0.97 (0.01)C. humilis Cu-polluted Takao 0.78 (0.14) 0.26 (0.04) 3.00 (0.05) 0.30 (0.05) 0.96 (0.02)C. humilis Cu-polluted Nagoya-1 1.78 (0.17) 0.62 (0.07) 2.88 (0.05) 0.59 (0.05) 0.94 (0.01)C. humilis Cu-polluted Nagoya-2 1.78 (0.30) 0.65 (0.11) 2.76 (0.02) 0.60 (0.10) 0.89 (0.02)C. humilis Cu-polluted Ueno 0.87 (–) 0.28 (–) 3.06 (–) 0.34 (–) 1.00 (–)C. humilis Cu-polluted Isehara-3 1.38 (–) 0.45 (–) 3.07 (–) 0.50 (–) 1.04 (–)C. humilis Cu-polluted Tsukuba-2 1.23 (–) 0.40 (–) 3.08 (–) 0.40 (–) 0.95 (–)

Fig. 3. Relative concentrations of secondary metabolites in S. japonicum (a) and C. humilis (b). Error bars represent standard deviations (n¼3). The values of the S. japonicumcontrol samples are averages (n¼2).

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control sites (3.0270.25, 1.6970.48, and 0.5570.12, respectively,for 12 samples at 4 control sites). However, the concentration oftotal carotenoids and the A435/A415 ratio of the Cu-polluted samples(0.4870.14 and 0.9570.05, respectively) were less (po0.05 andpo0.01, respectively) than those of the control samples (0.5870.12and 1.0370.09, respectively).

3.5. Analysis of lichen secondary metabolites

Fig. 3a shows the relative concentrations of secondary meta-bolites (atranorin, stictic acid, and norstictic acid) in S. japonicumgrowing in the Cu-polluted and control sites. The relative con-centration of each secondary metabolite in the Cu-polluted sam-ples was lower than that in the control samples. In both the Cu-polluted and control samples, however, the relative concentrationof norstictic acid is about one order of magnitude lower than therelative concentrations of atranorin and stictic acid.

On the other hand, the relative concentrations of secondarymetabolites (atranorin and fumarprotocetraric acid) in C. humilisgrowing in the Cu-polluted sites agreed with those in C. humilisgrowing in the control sites (Fig. 3b). Moreover, the ratio of theconcentrations of fumarprotocetraric acid and atranorin in theCu-polluted samples (0.5370.03) agreed with that in the controlsamples (0.5670.11).

4. Discussion

Results of the metal analysis indicate that S. japonicum and C.humilis are Cu-hyperaccumulating lichens. Although S. japonicum

is a common species in Japan (Yoshimura, 1974), this is the firstreport indicating that it is a Cu-hyperaccumulator. On the otherhand, Cu-hyperaccumulation in C. humilis was suggested in ourprevious study (Nakajima et al., 2012), and this finding wasconfirmed in the present study. The Cu concentrations in S.japonicum and C. humilis (1297 and 613 ppm, respectively;Table 1) growing under Cu-roofed buildings in low mountainsand urban areas are of the same order of magnitude as that in C.subconistea growing near an old mine (1.01�103 ppm) (Nakajimaet al., 2012). Hence, we found that Cu-hyperaccumulating lichenscan occur under Cu-roofed buildings in low mountain areas andeven in urban areas. Most such Cu-polluted sites in Japan have notyet been investigated; thus, future studies should find other Cu-hyperaccumulating lichens.

We found that the ratios of Al and Fe concentrations (Al/Feratios) in the lichen samples agree with each other and areindependent of the Cu concentration. The high level of correlationbetween Al and Fe concentrations in C. humilis (R¼0.974, po0.01)indicates that the Al/Fe ratios are almost constant (Fig. 4a). The Al/Feratio in C. humilis growing in Cu-polluted sites was 1.1070.29,which agrees with that in C. humilis growing in a control site (1.24)(Nakajima et al., 2012). The Al/Fe ratio was independent of Cuconcentration (R¼0.063). On the other hand, the Al/Fe ratios inS. japonicum growing in the Cu-polluted and control sites (1.11 and1.21, respectively) agree with that in C. humilis. Moreover, in apreliminary study, we obtained the same Al/Fe ratios in Stereo-caulon species growing in Cu-polluted and control sites (1.22 and1.24). Accordingly, we observed a perfect correlation (R¼1.000,po0.01) between the Al and Fe concentrations in Stereocaulon

Fig. 4. Correlations between Al and Fe concentrations in C. humilis (R¼0.974,po0.01) (a) and Stereocaulon species (R¼1.000, po0.01) (b). The solid lines arelinear regression lines.

Fig. 5. Correlations between the concentrations of Al and the pigments chlorophyll(chl.) a (R¼�0.905, po0.05) (a), chl. b (R¼�0.940, po0.01) (b), and totalcarotenoids (X+C) (R¼�0.926, po0.01) (c) in C. humilis. The solid lines are linearregression lines.

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species (Fig. 4b). Thus, we found that the Al/Fe ratio is commonbetween Cu-polluted and control samples of C. humilis andStereocaulon species. These correlations could be due to thepresence of small mineral particles or aerosols with the Al/Fe ratiotrapped by lichens. High correlations between Al and Fe concen-trations in soil and aerosols have been observed, which can beused to identify the origins of the soil and aerosols (Sheppardet al., 2000; Akata et al., 2007). Hence, it is likely that thecorrelations between Al and Fe concentrations in C. humilis andStereocaulon species are due to the presence of microcontaminantswith the Al/Fe ratio trapped by the lichens. A correlation betweenFe and Ti concentrations in Cladonia species is explained by asimilar phenomenon (Nieboer et al., 1978). This is consistent withour above result if the Al, Fe, and Ti concentrations are correlated.Correlations between the concentrations of these metals wereobserved in soil samples in Japan (Akata et al., 2007).

Results of the absorption spectra and pigment analysis showthat the chlorophyll a/b ratio of S. japonicum growing in the Cu-polluted sites was less than that of S. japonicum growing in thecontrol sites. Moreover, the difference in absorbance at 387 and407 nm, the concentration of total carotenoids, and the A435/A415

ratio of C. humilis growing in the Cu-polluted sites were less thanthose of C. humilis growing in the control sites, suggesting thatthese values are decreased by Cu stress. To confirm this possibility,correlation coefficients and p-values between Cu concentrationand these values were calculated in C. humilis; however, nocorrelation was observed. This indicates that the effect of Cu stresson pigments and spectral properties is neither simple nor direct.We unexpectedly found high negative correlations between theconcentrations of Al and Fe and those of chlorophyll a, b, and totalcarotenoids (Ro�0.905, po0.05 for Al and Ro�0.862, po0.05for Fe). Fig. 5 shows the correlations between Al and pigment

concentrations in C. humilis. Similar correlations were observedbetween Fe and pigment concentrations. This is expected because,as discussed in the previous paragraph, the Al concentration ishighly correlated with the Fe concentration. Furthermore, weobserved similar negative correlations between the concentrationsof the 2 metals and the 3 pigments in the 4 samples of Stereo-caulon species (R¼�0.991, po0.01 and R¼�0.983, po0.05 forthe Al concentration and chlorophyll a or b concentration, respec-tively). The high negative correlations between Al and pigmentconcentrations could be caused by Al stress in C. humilis andStereocaulon species. Al can substitute for Mg in biological systemsand inhibit the function of their enzymes (Shaw et al., 2010).Similarly, Al stress may reduce pigment concentrations.

H. Nakajima et al. / Ecotoxicology and Environmental Safety 97 (2013) 154–159 159

The concentrations of secondary metabolites in S. japonicumgrowing in the Cu-polluted site are likely to be less than those inS. japonicum growing in the control sites (Fig. 3a). More samples ofS. japonicum are needed to show a definite relationship betweenthe metabolite concentrations in Cu-polluted and control samples.On the other hand, the concentrations of secondary metabolites inC. humilis growing in the Cu-polluted sites agree with those in thecontrol sites (Fig. 3b). This indicates that the metabolite concen-trations are not influenced by Cu stress.

5. Conclusions

We investigated lichens near temple and shrine buildings withCu roofs in Japan and found that S. japonicum and C. humilisgrowing in the Cu-polluted sites are Cu-hyperaccumulatinglichens. Differences in pigment concentrations and pigmentabsorption spectra were observed between the Cu-polluted andcontrol samples of S. japonicum and C. humilis. However, nocorrelation was found between Cu and pigment concentrations.We observed a positive correlation between Al and Fe concentra-tions and unexpectedly found high negative correlations betweenAl and pigment concentrations. This suggests that Al stressreduces pigment concentrations. The concentrations of secondarymetabolites in C. humilis growing in the Cu-polluted site agreedwith those in the control sites. This indicates that the metaboliteconcentrations are independent of Cu stress. Through this work,we have gained a deeper understanding of the ecotoxicology ofthe Cu-hyperaccumulating lichens S. japonicum and C. humilis.

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