Effect of metallic pollutants on the physiology of lichen,Pyxine subcinerea Stirton in Garhwal Himalayas

Vertika Shukla & Dalip K. Upreti

Received: 8 November 2006 /Accepted: 17 August 2007 /Published online: 19 September 2007# Springer Science + Business Media B.V. 2007

Abstract Chl. a, Chl. b, total Chl., Carotenoid,Protein and OD 435/OD 415 ratio were measured toestimate the possible damage caused by the metallicpollutants in the lichen, Pyxine subcinerea Stirtoncollected from four different sites of SrinagarGarhwal, Uttaranchal, India. Multiple correlationanalysis revealed significant correlation (P<0.001)among the Fe, Ni, Cu, Cr, Zn and Pb metals analysed.Cd did not correlate with any other metals except Fe(P<0.05). Cu, Pb and Zn, are the main constituents ofthe vehicular emissions had significant positivecorrelation (P<0.001) with protein content while,the OD 435/OD 415 ratio values decreased statisti-cally (P<0.001) with increase in amount of Cu, Pband Zn.

Keywords Garhwal Himalayas . India . Lichen .

Metal-physiology interaction . Pyxine subcinerea

Introduction

Metallic pollutants are known to disrupt the vitalphysiological processes. Heavy metals react with thephotosynthetic apparatus at various levels of organi-zation and causes alteration of the functions ofchloroplast membrane, particularly PS II and PS I,including delayed Chlorophyll degradation, decreasein total chlrophyll and Chl.a/b ratio (Bishnoi et al.1993; Sheoran 1990). Because of the lower contribu-tion of algae (about 5%) in the constitution of thelichen thallus, slight variation in the ambient atmo-sphere is reflected by the change in the physiology oflichen. This feature of lichen has been extensivelyutilized to assess the impact of heavy metal on thephysiological parameters, like net photosynthesis(Boonpragob and Nash 1991; von Arb 1987), chlo-rophyll degradation (Garty et al. 1985, 1992),chlorophyll content and spectral reflectance response(Garty et al. 1997a,b), photosynthesis along withprotein synthesis (von Arb et al. 1990) and ATP(Kardish et al. 1987). The objective of the presentinvestigation is to statistically correlate the elementalbioaccumulation, by the lichen thalli, with thephysiological responses to interpret the probablerelationship between biological damage and the metalpollutant and provide evidence for the usefulness ofsome physiological parameters as indicator of metallicpollution. This is for the first time in India an effort

Environ Monit Assess (2008) 141:237–243DOI 10.1007/s10661-007-9891-z

V. Shukla :D. K. Upreti (*)Lichenology Laboratory,National Botanical Research Institute,Rana Pratap Marg, P. B. No. 436, Lucknow 226 001, Indiae-mail: upretidk@rediffmail.com

has made to statistically correlate the physiology oflichens with the ambient metallic pollution and alsothe utility of Pyxine subcinerea as a biomonitoringdevice.

Study area

Uttaranchal, a hilly state, comprises 17.3% of India’stotal land area with 51,125 km2. It has a population ofabout 6.0 million. The study area (Fig. 1), between29° 20′ N-30° 15′ N latitude and 78° 10′ E-79° 20′ Elongitude. Srinagar is situated in a sprawling valleyon the bank of Alaknanda river in the CentralGarhwal Himalayas. The area of the Srinagar city isabout 9.7 km2, total population census is 18,791, andit is the location of Garhwal University. The locationof the city (located on the National Highway 58,Delhi-Niti Pass, strategically important) and rapidurbanization taking place in the city provides anoptimal site to investigate the impact of increasing

traffic activity and urbanization on the Himalayanecosystem. The lichen samples, collected fromSrinagar area, were from the junction of roads fromKotdwar, Rishikesh, Tehri-Garhwal, Kedarnath andBadrinath which serves as a main halting point for thepilgrims of ‘Char Dham Yatra’.

Material and methods material

The study was carried out with the foliose lichen P.subcinerea Stirton, growing in their natural habitatfrom 4 different sites, on trees of Melia between 0.5–1.5 m from the ground in Pauri and Srinagar(Garhwal), Uttaranchal Himalayas, India, in June2005 (Table 1). The epiphytic lichen, P. subcinereais especially well suited for use in physiologicalstudies under stressed condition as grows luxuriantly(naturally) at both polluted and non-polluted sites.Approximately 2–3 g of thallus (of similar size up to3 cm) was taken from each site. Samples were

Fig. 1 Map showing collection sites in Srinagar (Garhwal), Uttaranchal, India

238 Environ Monit Assess (2008) 141:237–243

collected in triplicate. The size of thalli varied fromsite to site. Most of the samples were collected fromthe side of the trees facing towards the road.

Analysis of the integrity of the photobiont chlorophyllin the lichen thallus

The method developed by Ronen and Galun (1984)was used to measure integrity of the photobiopntchlorophyll. Subsamples of 20 mg each was used.The chlorophyll was extracted overnight in the dark in5 ml dimethyl sulfoxide (DMSO, Merck, analyticalgrade). The ratio of chlorophyll a to phaeophytin a(OD 435 nm/OD 415 nm ratio) was determined usinga Genesys 10 uv scanning spectrophotometer.

Pigment analysis

Photosynthetic pigments (chlorophyll a, chlorophyll band carotenoid) were extracted into 80% acetone

(Merck, Analytical grade) and their concentrationsdetermined using standard spectrophotometric proce-dures. Pigment extractions were performed by grind-ing samples with acid washed sand 50 mg magnesiumcarbonate and 10 ml acetone (80%) on ice in dimlight. The slurry was transferred to a 10 ml centrifugetube shaken vigorously and spun at 10,000 rpm for5 min. The supernatant was then decanted, kept incold and dark, and the pellet resuspended in 1.5 mlacetone (80%) and centrifuged as above. The super-natant were then combined, made to a known volumeand analysed using Genesys 10 uv scanning spectro-photometer.

The chlorophyll content was calculated fromabsorbance values at 663 and 645 nm according tothe equation of Arnon (1949, p. 1). The totalcarotenoid content was calculated according toParsons et al. (1984) from absorbance values at480 and 510 nm using Genesys 10 uv scanningspectrophotometer.

Table 2 Physiological measurements in thalli of P. subcinerea collected from various sites of Srinagar (Garhwal)

f ratio f probability Sites

Malli Kalikamali Srinagar Road Government InterCollege (GIC)

Chlorophyll a 257* 0.0 0.0111±0.0005 0.0113±0.0005 0.19±0.019 0.013±0.001Chlorophyll b 115* 0.0 0.0069±0.0004 0.0059±0.0002 0.0074±0.0002 0.0052±0.0005Total Chlorophyll 491* 0.0 0.0181±0.0002 0.0171±0.0003 0.0267±0.0005 0.0182±0.0002OD 435/OD 415 ratio 7165* 0.0 0.876±0.003 1.114±0.001 1.046±0.003 0.989±0.002Carotenoid 666* 0.0 0.673±0.001 0.490±0.02 0.473±0.001 0.3033±0.002Protein 1.95 0.2 46.03±7.74 30.99±6.74 33.06±9.94 32.99±7.37

Concentration are given as μg g−1 f. wt. (values expressed as mean ± standard error) and f values along with f probability obtained byone way ANOVA analysis

*P<0.01

Table 1 Description of the sites selection for the collection of lichen specimen in the city of Srinagar (Garhwal), Uttaranchal, India

S. No. Sites Vehicular pollution Human interference Remarks

1 Malli 500 v/hr (mostly heavyvehicles)

Road is under construction Highlypolluted

2 Kalikamalidharmshala

270 v/hr (heavy + lightvehicles)

Site is having dense tree canopy with maximum humaninterference

Moderatelypolluted

3 Srinagar Road 180 v/hr (mostly lightvehicles)

Construction of buildings is maximum on this road Moderatelypolluted

4 G.I.C., Srinagar 65 v/hr (mostly lightvehicles)

Site is located on the non metallic road on a hill top just abovethe Srinagar bus stand

Moderatelypolluted

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Protein estimation

Protein was estimated using Lowry method (Lowryet al. 1951) using Folin phenol as reagent andcalculations were made from absorbance values at700 nm.

Metal analysis

The lichen thalli (approximately 4 gm) were removedfrom the bark with sharp knife. The samples wereoven dried for 12 h to a constant weight at 90°C. Thedried lichen samples (3 replicates) were grinded topowder (1.0 g each) and digested in mixture ofconcentrated HNO3 and HClO4 (V/V 9:1) for 1 h.Residues were filtered through Whatman Filter paperNo. 42 and diluted to 15 ml with double distilledwater. Analysis was done with Flame Atomic

Absorption Spectrophotometer (Perkin Elmer, modelA Analyst 300). Stock standards were from MerckIndia and traceable to NIST (National Institute ofStandards Technology). Working standards wereprepared from the stock using deionised water fordilution.

Statistical analysis was performed using softwareprogram, INDOSTAT. One way analysis of variance(ANOVA) and correlation matrix tables were createdto test the variability and correlation of the dataanalysed.

Result and discussion

The results of the physiological (Table 2) and elementalanalysis (Table 3) were evaluated by one-wayANOVA, thus evaluating the effects of the sampling

Table 3 Mineral element concentrations in thalli of P. subcinerea collected from various sites of Srinagar (Garhwal)

Metals f ratio f probability Sites

Malli Kalikamali Srinagar Road Government InterCollege (GIC)

Fe 0.35 0.79 55.93±2.26 43.08±12.82 54.53±7.74 46.01±13.8Ni 5.6* 0.03 0.1407±0.0053 0.0200±0.0058 0.141±0.0576 0.020±0.058Cu 7.65* 0.02 0.849±0.0013 0.273±0.1047 0.531±0.1188 0.344±0.059Cr 4.77* 0.05 0.198±0.0188 0.065±0.0036 0.162±0.0576 0.070±0.0412Cd 1.21 0.38 0.012±0.0385 0.008±0.001 0.014±0.0012 0.013±0.0038Pb 4.12 0.07 0.622±0.1179 0.262±0.060 0.456±0.0585 0.351±0.0112Zn 9.41* 0.01 4.69±0.610 1.76±0.3817 3.68±0.8588 1.93±0.2517

Concentration are given as μg g−1 d. wt. (values expressed as mean±standard error) and f values along with f probability obtained byone way ANOVA analysis

*P<0.05

Table 4 Values of correlation matrix between the amounts of some elements found in P. subcinerea, and between these elements andphysiological parameter studied (Total Chl., Carotenoid, Protein and OD 435/OD 415 ratio)

Fe Ni Cu Cr Cd Pb Zn Total Chl. OD 435/OD 415 ratio

Protein Carotenoid

Fe j 0.978*** 0.904*** 0.979*** 0.647* 0.933*** 0.970*** 0.5468 −0.665* 0.709** 0.612*Ni j 0.855*** 0.975*** 0.531 0.867*** 0.955*** 0.6140* −0.517 0.630* 0.673*Cu j 0.949*** 0.409 0.990*** 0.969*** 0.1400 −0.848*** 0.941*** 0.792**Cr j 0.483 0.950*** 0.997*** 0.4272 −0.673* 0.788** 0.761**Cd j 0.532 0.469 0.6158* −0.498 0.221 −0.193Pb j 0.968*** 0.2125 −0.870*** 0.911*** 0.703*Zn j 0.3635 −0.720** 0.831*** 0.776**

*P<0.05; **P<0.01; ***P<0.001

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site on the bioaccumulation and the physiologicalstatus of the photobiont. The data are expressed as Fratio and F probability values. The contaminationlevels of Ni, Cu, Cr and Zn are significantly different(P<0.05) from each other (between the sites). Physi-ological parameters except protein, significantly varyamong the sites (P<0.01).

Among the four sites studied Malli, located onSrinagar-Pauri Road had maximum concentration ofFe, Ni, Cu, Cr, Pb and Zn at 55.93, 0.1407, 0.8490,0.1980, 0.6220 and 4.69 μg g−1 respectively, followedby PWD Guest house. The total metal concentrationranged from 62.44–45.47 μg g−1 with an averageconcentration of 52.74 μg g−1.

The correlation coefficient were calculated (Table 4)for concentration in paired element and for the elementcontent in the lichen and the physiological parametersat the different sites. The possible source of elementmay be indicated by significant correlation betweenelements in the lichen thallus. The correlations (allsignificant at P<0.001) of Cu and Fe, Cu and Zn, Feand Zn, Pb and Zn, Pb and Fe and Pb and Ni indicatesmotor vehicles as possible originators. Ormrod (1984,p. 291) has linked automobile traffic in Los Angeleswith the release of Pb, Zn, Cd, and Cu. Cadmium isreleased by wearing of motorcar tires. Lubricating oiloften contains Cd, Cu, and Zn. Zn may also be emittedby automobile tires, brake pads (Ward 1989). Nickel isfound in car metal plating in welded plates (Ward1989) and in tires (Sadiq et al. 1989)

From the Table 4 it is clear that the thallus contentof all the metal analysed is inversely correlated to theOD 435/OD 415 ratio while directly related to theprotein and carotenoid content. Lead and copperhaving correlation values, r=−0.8703 and −0.8485respectively shows negative correlation (P<0.001)

with OD 435/OD 415 ratio. Garty et al. (1985, p. 67)have also observed similar results in the Ramalinaduriaei transplanted at 13 biomonitoring sites, inIsrael, for one year.

Carotenoid has significant positive correlation withCu, Cr, and Zn (P<0.01), while shows negativecorrelation with cadmium (r=−0.1928). Carotenoidcontent has maximum positive correlation withcopper (r=0.7924). It indicates that copper in elevatedconcentrations may supplement synthesis of caroten-oid content, which is in accordance with the findingsof Pawlik-Skowrońska et al. (2006, p. 267). Copperin high concentration can decrease total carotenoidconcentration in Trebouxia cell (Bačkor et al. 2003),the known photobiont of the family Physciaceae,tolerant species shows no alteration in the totalcarotenoid content.

Nickel (r=0.6140) and Cadmium (r=0.6158) haspositive correlation (P<0.05) with total chlorophyll.This indicates that under stressed condition, in naturalhabitat, the total chlorophyll increases. von Arb(1987, p. 343) and von Arb et al. (1990, p. 431) havereported increase in total chlorophyll in the lichenspecies, Parmelia sulcata under polluted conditions.

Heavy metals are known to alter the biosynthesisof protein in higher plants (Neumann et al. 1994). In arecent study on Phaeophyscia hispidula, growing inits natural habitat (Shukla and Upreti 2007) increasedair pollution has been reported to cause increase inprotein synthesis in order to combat the negativeimpact of increased elemental concentration on thelichen thalli. Protein levels are highly positivelycorrelated at 0.001 level with Cu, Pb, and Zn.

Various interactions are known to occur whenplants are exposed to unfavorable concentrations ofmore than one trace element. Such combination

Table 5 Values of coefficient of rank correlation between the various physiological parameters (Chl. a, Chl. b, Total Chl., OD 435/OD 415 ratio, Carotenoid and Protein)

Chl. a Chl. b Total chlorophyll OD 435/OD 415 ratio Carotenoid Protein

Chl. A j 0.715* 0.994** 0.264 −0.061 −0.265Chl. B j 0.718* −0.194 0.650 0.389Total chlorophyll j 0.172 −0.062 −0.202OD 435/OD 415 ratio j −0.466 −0.915**Carotenoid j 0.779*

*P<0.05; **P<0.01

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effects were categorized by Berry and Wallace (1981,p. 13) as independent, additive, synergistic orantagonistic.

Correlation of various physiological parameters(Tables 2 and 5) shows significant correlation of totalchl. content with chl. a (P<0.01) and chl. b (P<0.05)while carotenoid and protein had negative correlationwith chl. a, total chl. and OD 435/OD 415. Proteinhad highly significant positive correlation value ofr=0.7799 with carotenoid. The negative correlationof protein with OD 435/OD 415 ratio(r=−0.9152;P<0.01) affirms that under stressed condition theintegrity of chlorophyll decreases, protein contentincreases significantly. Increase in protein due tostressed condition has been reported in higher plantsby Neumann et al. (1994, p. 360).

The results show a clear correlation between themetallic pollutants and some physiological parametersof P. subcinerea, which represents the effect ofmetallic pollutants on the metabolic process of thespecies. The high correlation of OD 435/OD 415 ratiowith metallic pollutants is consistent with studiessimilar to Garty et al. (1985, 1992). Among the sixmetals analysed, Cu, Pb and Zn seems to causeextensive damage to the biological apparatus bycausing alteration in the vital physiological processlike chlorophyll content and protein. Thus chlorophyllcontent, OD 435/OD 415 ratio and protein contentappears to be the appropriate parameter to monitor airpollution in large area because P. subcinerea, apollution tolerant species has wide geographicaldistribution in the Garhwal Himalayas of India.

Conclusion

The clearly indicate that Cu, Pb, and Zn significantlyaffects the physiology of lichen, P. subcinerea. Thisstudy provides the baseline data for the futurebiomonitoring studies to be carried out to observethe changing air quality of the Himalayan biospheredue to deforestation and urbanization. The utility ofP. subcinerea Stiton as a biomonitoring device hasalso been reported for the first time.

Acknowledgements We are grateful to Dr. Rakesh K. Tuli,Director, National Botanical Research Institute, Lucknow forproviding laboratory facilities; to Dr. Sudhir Shukla for help in

statistical analysis and to the Science and Society Division,Department of Science and Technology, New Delhi forfinancial support (SSD/SS/063/2003).

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