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Summer Research Fellowship Programme of India's Science Academies

Biomonitoring study using foliose lichen Pyxine cocoes

Ms. Josepheena Joseph

Department of Plant Sciences, Central University of Punjab, Bathinda 151 001, Punjab

Dr. Dalip Kumar Upreti

CSIR- Emeritus Scientist, Lichenology Laboratory, CSIR- National Botanical Research Institute, Uttar Pradesh, Lucknow 226 001

Abstract

In the recent years, a number of biomonitoring assessment studies are carried out in different regions of the world, utilizing lichen species/ communities. Among different growth forms of lichen, the foliose lichen genera viz., Phaeophyscia and Pyxine which grow abundantly in tropical region are used for biomonitoring studies frequently. The present investigation initiated with an aim to understand the physiological changes occurred in transplanted Pyxine cocoes with relation to low and high vehicular activity at two different road sides. The changes in photosynthetic pigments, protein content and heavy metal were examined using transplantation method for a fortnight time period. The result shows an increment in protein content in transplants as a result of stress faced by lichen due to vehicular emissions. In contrast, heavy metal gets decremented due to heavy precipitation during the transplantation period. It is evident from the experiment that lichen can be used for biomonitoring study due to its unique characteristic features.

Keywords: lichen, biomonitoring, heavy metal, sensitivity, tolerance

Abbreviations

Abbreviations
IPCC Intergovernmental Panel on Climatic Change 
ICP MS  Inductively Coupled Plasma Mass Spectrometry
 PAHsPolycyclic Aromatic Hydrocarbons 
BSA Bovine Serum Albumin 
MDA Malondialdehyde 
 HPCDHydroperoxy Conjugated Dienes 
 ACC 1- Aminocyclopropane-1-Carboxylic Acid

 INTRODUCTION

Climate change is considered to be the most concerned and challenging topic of the present world. IPCC (Intergovernmental Panel on Climatic Change) assures that global climate is changing in an alarming rate (IPCC, 2007). Thus, determination of air quality within a natural ecosystem becomes an inevitable parameter. Lichens, a symbiotic association comprises of both fungal as well as algal component, are considered to be an excellent organism for monitoring environmental pollution together with climatic changes in an area. The sensitivity of lichens towards air pollution; good ability for bioaccumulation of both organic, inorganic chemical compounds and radionuclides are the major characteristic features which make them excellent organism for biomonitoring environment and climatic changes of an area. Other morphological and anatomical features such as lack of vascular system, absence of root system, lack of cuticle as well as stomata enable them to depend up on the atmospheric sources for the nutrients. Lichens show differential sensitivity towards specific air pollutants. Some are sensitive whereas, some are tolerant to high levels of pollutants as well as contaminants. These features enable lichens to be used as an indicator species (i.e., sensitivity and tolerance).

Biomonitoring is more recommendable since, it is based on the quantitative estimation in terms of physical as well as chemical aspects over a period of time. Thus, provides, an accurate data regarding atmospheric pollutants and the subsequent contamination over an area. The essential characteristic features for using lichen as a biomonitor includes:

(1) it must accumulate measurable amount of metals in their entire thallus

(2) it must be perennial i.e., should available throughout the year

(3) cost of collection and analysis should be acceptable

(4) the study must be repeatable

(5) abundance of the sample in the study area

(6) tolerance of pollutants at relevant levels

In context to India, foliose lichen Pyxine cocoes which grows most abundantly both in temperate as well as tropical region were frequently used for carrying out biomonitoring studies. Thus, the project aims to determine the heavy metal accumulation as well as estimation of chlorophyll, proteins and carotenoids in both controlled and transplanted samples. The change in the level of metals accumulated in transplanted and controlled lichen samples together with estimation of chlorophyll, protein and carotenoids will provide an account of pollutant load in the area under study.

 OBJECTIVE

To study the biomonitoring ability of lichen by using the foliose lichen genus Pyxine cocoes.

Review of Literature

Pollutant accumulation in lichens

Apart from inorganic metals (trace and heavy metals) lichen has an ability to accumulate both organic and radionuclides. PAHs (Polycyclic Aromatic Hydrocarbons), are compounds, which are synthesized by the incomplete combustion of organic material both natural as well as manmade activity. PAHs are formed mainly in three ways: a) due to high temperature pyrolysis of organic material; b) low to moderate temperature diagenesis of sedimentary organic material to form fossil fuel; c) direct biosynthesis by microbes and plants (Ravindra et al., 2008). Moreover, forest fires and agricultural burning also contributes for the PAHs formation and these are then absorbed with the suspended particle in the atmosphere and eventually enter terrestrial as well as aquatic ecosystem (Baumard et al., 1998).

Due to persistent nature, PAHs have the ability to get transported to long distances which is far away from the origin through process called volatilization and condensation, the process precisely termed as hoping (Fernandez et al., 1999). PAHs attracted the attention of research, due to its role as carcinogens. Among PAHs, 5-6 ringed are known to be potential carcinogens. Benzopyrene, one among them considered to be the most dreadful in causing cancer (Part et al., 2002).

In India, PAHs accumulation studies with the aid of lichen recently initiate in the Himalayan region of Uttarakhand (Shukla & Upreti, 2009). The mechanism of bioaccumulation of PAHs is attributed to the donor acceptor complex mechanism which in turn are examined to be formed between PAHs and compounds of biological importance (Harvey & Halonen, 1968). PAHs which are hydrophobic in nature readily combines with the organic moiety to from an adduct. The higher accumulation of 2, 3 ring of PAHs in lichen may be because, most of the species containing secondary metabolites is due to the presence of despides and desposones which have -OH as active sites which then further facilitate adduct formation. Pyxine as well as Phaephyscia possess skyrin triterpene and lichexanthone (also contain -OH active site) which readily combines with most of the PAHs and hence accumulates on the lichen thallus (Shukla et al., 2014).

The radionuclides are trace elements which are mostly found in rocks in varying concentrations. The radionuclides such as Uranium-238 and Thorium-232 release large amount of energy in the form of alpha, beta and gamma radiations. These on striking with the living organism disrupts the cell wall as well as its cell integrity which result in mutation and further cancer and other health related problems. Man-made source of radionuclides is mostly released due to nuclear accidents. The Chernobyl accident in 1986, explosion of the deposit of nuclear waste in kyshtym in Eastern Urals in 1957 and the Fukushima nuclear disaster in the year 2011 are the dreadful among them. Number of studies carried out before and after the accidents revealed that lichens and mosses are able to accumulate high amount of radioactivity (Eckl et al., 1986). Moreover, lichens are used frequently to monitor spatial pattern in radioactive deposition over wide areas (Feige et al., 1990).

Lichen especially, the foliose growth form has reported to possess high surface to mass ratio, due to which it accumulates heavy metals and radionuclides effectively in the thalli (Seaward et al., 1988; Nimis et al., 1993). It is also found that the radioceasium ions get strongly bound to the lichen thallus and thus difficult to remove, this explains that the ions which get accumulated on the lichen thalli further gets bounded to cytoplasmic molecules through process called active translocation (Subbotina et. al, 1961). It is also reported that lichen is more resistant to high radioactivity than other organism. The lichens are considered to be the living accumulators of natural as well as manmade radionuclides and heavy metals (Eckl et al., 1986).

The heavy metals are chemical elements with a specific gravity i.e., at least five times the specific gravity of water. The heavy metals are then categorized in to three classes. They are class A, these are oxygen seeking elements and class B which are N/S seeking metals and the third termed as the borderline metals. Road dust are mostly produced from non exhaust vehicular emissions which are generated due to tire, brake, clutch wear, road surface wear and other vehicle and road component degradation (Adamiec et al., 2016). These road dust includes various components like heavy metals such as Cd, Cr, Cu, Ni, Pb, Zn, Fe, Se, Ba, Ti and Pd which are then analyzed by HPLC ICP MS. Model No:7500cx.

Heavy metals from vehicular emissions act as a threat to human as well as environment because of its adverse effect on ecosystem in terms of polluting air, water and soil. The dust from the vehicle are generated from number of sources such as wearing of breaking system, tires, clutch plates, erosion of the active layer of the catalytic convertor etc. The main contributor for the road dust contamination are the tire wear and road surface abrasion, many experiments related to the tire wear dust has been examined and the studies shows that these dust comprises of Zn, Cd, Co, Cr, Cu, Hg, Mo, Ni and Pb. Among this, Zn considered to be the abundant heavy metal released from tire wear (Fukuzaki et al., 1986). In accordance with Ozaki et al. (2004) tire comprise of 1.3–1.7% Zn. The study shows that tire wear emissions range between 16 and 90 mg/tire/km (Baekken, 1993; Lee et al., 1997). One exciting finding shows that an average mass of new car tire is approximately 8 kg but during, its lifetime it loses up to 1.5 kg. This reveals that within 3 years about 10–20% of rubber enters the environment due to abrasion. The greatest wear of tire occurs during acceleration, breaking and cornering (Adamiec et al., 2016). It is also reported that heavy metal accumulation is affected by both vehicle operation as well as road abrasion. It is noted that more tire abrasion occurs when vehicle move through a concrete motorway than that of asphalt surface (Duong & Lee, 2011).

Moreover, driving on concrete surface also need high energy use which in turn results in high fuel consumption. Heavy metal concentration in road dust also strongly depends on vehicular speed, higher speed in turn results in greater tire wire and increased fuel combustion. Another source of road dust is from braking system i.e., during rapid braking, brakes are exposed to extensive heat from friction which is transmitted to the brake disc which then results in the emission of particles. The most intense brake wear occurs at intersections, corners, traffic lights and through forced braking and also it is reported that brake dust contains not only Fe but also significant amount of Cu, Sb, Ba, Al, Si, S, Ti, Zn, Ni, Cr, Pb and small amount of Cd (Adamiec et al., 2016).

Mechanism of accumulation

The secondary metabolites present in lichen thalli play an important role in metal uptake. All the secondary metabolites are fungal in origin, they are formed from primary metabolites but don’t have direct involvement in metabolism, these are mostly deposited on the surface of hyphae than within cells. Therefore, these are termed as extracellular compounds (Karakoti et al., 2014). Lichens produce more than 1000 secondary metabolites, each have their own characteristic chemicals (Nash, 1996; Shukla et al., 2014). The secondary metabolites are mostly produced from three main pathways i.e., acetyl-polymalonyl pathway, mevalonic acid pathway and shikimic acid pathway (Boustie & Grube, 2005). Lichen substances such as despides and desposones which are having lone pair of electrons and number of hydroxyl groups provide an inherent characteristic of chelating property. Adsorption of metals mainly depend on chemical nature of lichen substance as well as the electron available in the metal ion for binding (Hauk & Huneck, 2007). The lichen substances are known to function in vitro as chelators of cation including heavy metals (Purvis et al., 1987). It is reported that cations including heavy metal can bind to extracellular sites of mycobiont and photobiont cell walls. The cell wall contains sites for metal accumulation due to the presence of negatively charged anionic sites (Collins & Farrar, 1978). These anionic sites are contributed by carboxyl, phosphate, amine and hydroxyl groups. Metal accumulation is always accompanied by rapid release of protons (Nieboer et al., 1976).

Heavy metal accumulation is considered to be very dynamic process. Transplantation studies using lichen samples reveals that most lichen respond to heavy metal accumulation with in few months. The atmospheric pollutants are deposited on the lichen surface or gets entrapped between the intercellular spaces of the medulla (Garty et al., 1979). Lichens are able to retain as well as accumulate heavy metals more than their physiological requirement and can tolerate high metal concentration by sequestering metals extracellularly as oxalate crystals complexing with the lichen acids. Most of the metals deposited are immobile. Thus, it gets transported only through chelation as well as sequestration mechanism (Backor et al., 2009).

The accumulation of heavy metals on the lichen thalli depend on many factors such as availability of elements, the characteristics of the plant such as species, age, state of health and type of reproduction. The other parameters such as temperature, moisture, substratum features also influence the accumulation of heavy metals (Baker, 1983). The mechanism of accumulation of heavy metals are explained by three ways. These are: 1) extracellular ion exchange process; 2) intracellular accumulation; 3) entrapment of particles (Richardson, 1995). Ion exchange process has been explained by submerging lichen thallus in an electrolyte solution, and noted that they can remove certain metals such as Ca²⁺ and Pb²⁺, thus, the experiment shows that most of the accumulated metals in the lichen thallus occurs in exchangeable form. The exposed surface of submerged lichen thalli is differentiated in to four main zones such as Zone A, Zone B, Zone C and Zone D (Fig.1). The zone A is referred to as ion exchange surface. The functional group binding cations and anions are fixed on the ion exchange surface i.e., hyphal cell wall and this has access to the water in the zone B, which in contact reacts with the metal ions. Hence, Zone B is termed as the reaction zone. The ions entering or leaving the Zone B diffuse across Zone C and then Zone D where, Zone D is termed as the bulk solution zone (Nieboer et al., 1978).

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    Exposed surface of submerged lichen thalli, Zone A, with functional group binding cations projecting to Zone B, adjacent lies Zone C and Zone D. (Redrawn from Nieboer et al. 1978)

    Intracellular uptake occurs very slow compared to the ion exchange process and the uptake increases with the time (Beckett & Brown, 1984). It is seen that intracellular uptake is a carrier mediated process with expenditure of energy, such expense of energy is seen in the uptake process of phosphate (Farrar, 1976). It is reported that mineral particulate trapping was considered to be significant accumulative pathway (Nieboer et al., 1978). Particular entrapment occurs due to the presence of intercellular spaces within the lichen thallus. These spaces entrap certain pollutants such as heavy metals (Nieboer et al., 1978).

    Structures aiding particle entrapment

    The amount of metal contained in a lichen is, species dependent and refers decisively to its morphological and structural features (Chiarenzelli et al., 1997). It is shown that foliose lichen can accumulate more metals than fruticose growth form due to its morphological features. The surface features such as cilia, pits, isidia, and rugosity enhance the efficiency of particle entrapment (Puckett & Finegan, 1980). Rhizinae as well as medulla plays an important role in metal accumulation and translocation especially through terricolous lichen. The experiment conducted by Peltigera canina and P. rufescens responds to metal pollution by reducing thallus size, rhizine length and also results in darkening of the thallus. Rhizinae plays an important role in absorbing, accumulating, translocating and regulating metals in Peltigera canina (Goyal & Seaward, 1982).

     Location of metals in lichen thallus

    It is seen that central portion of the thallus is enriched with the metals. Many factors influence the location of metals such as age and thalli size (Garty et al., 2013). It is seen that the metals such as Na⁺ and Cl⁻ localize in the mycobiont whereas, Fe²⁺ is associated with the algal cells and fungal hyphae. K⁺ was detected both in fungal as well as algal cells. Fe²⁺ also seems to accumulate in the upper cortex of lichen. Zn²⁺ seems to occur in the fungal cell walls and in the protoplasm of the mycobiont. Cu²⁺ found to be restricted to the apothecial surface and hypothecium of Lecidellla bullata (Purvis, 1984). Ca²⁺ accumulate mainly in medulla of certain lichens such as Acarospora rugulosa and Lecidea theides (Purvis, 1984). Ca²⁺ seems to accumulate in medulla and in the cortical hypha of the algal zone, whereas K⁺ accumulate mainly in the algal zone in presence of Mg²⁺ and P (Asta, 1992).

    Factors affecting accumulation

    Many factors affect the accumulation of heavy metal on lichen thallus. One such factor is the rainfall. The metals can be removed by rainfall or heavy precipitation, this dislodge the contaminants on the thallus surface (Brown & Brown, 1991). Therefore, the best season suitable for the growth and mineral uptake of is when lichen thalli is wet due to high mist or fog as in most of the temperate areas i.e., more precisely winter seems to be more preferable season for the best growth of lichen.

    The second factor is the time of exposure of lichen thallus to the pollutants. Mostly, short time exposure is more preferable because, after long time exposure the lichen sample get saturated with element, lose biomass and change surface structures and physiological performance or activity (Bargagli & Mikhailova, 2002). Surface structure, adhesiveness, and water holding capacity also affects the metal accumulation (Brown & Beckett, 1985). Since, heavy metal accumulation is a physicochemical process where, pH also plays an important role in the accumulation mechanism i.e., decrease in pH increases the solubility of metals which in turn increases the bioavailability of metals (Nieboer et al., 1976). Temperature is another important factor affecting the metal accumulation. The metal uptake increases with increasing temperature (Nieboer et al., 1976). Metal uptake also influenced by the location i.e., it varies based on altitude due to different amount of precipitation and deposition of more soluble elements, therefore the metal concentration increases with the altitude (Kral et al., 1989). Lichens growing on isolated trees accumulate more heavy metals than the dense region because more wind passes through (Bargagli & Mikhaiova, 2002). It is also noted that dead thalli accumulate more heavy metal than living thalli (Nieboer et al., 1978). Thus, it is well clear that metal accumulation is considered to be a very complex process involving many factors.

     Physiological changes due to accumulation of heavy metal

    There are a number of physiological changes associated with the accumulation of heavy metals. It is seen that much lower concentration of Zn and Cd results in decrement in the photosynthesis. Especially, the PSII complex affects due to the uptake of heavy metals such as Cu²⁺. In Ramalina fastigiata, it is seen that intracellular Cu²⁺ content results in the total inhibition of PSII photochemical reactions (Branquino et al., 1999). Heavy metal also affects the chlorophyll content and integrity, but not all metals contribute for degradation. The metals such as Cu, Hg, Ag ions seems to indulge with the integrity of chlorophyll pigment. Certain metals are directly linked with the disintegration as well as damage of chlorophyll synthesis (Garty et al., 2013). It is also seen that the degradation of chlorophyll increases with the indulgence of certain heavy metals such a Mg, Cr, Fe and Cd (Bartok et al., 1992). Cu plays an important role with the biosynthesis of chlorophyll as well as lipid peroxidation process in photosynthetic membranes (Chettri et al., 1998). Moreover, it is seen that as concentration of heavy metal increases, it also increases the free radical formation. Increase in heavy metal concentration also results in the increase of water loss (Chettri & Sawdis, 1997). The metal solution also contains inhibitory effect on germination of ascospores.

    Moreover, MDA (Malondialdehyde) is a highly reactive aldehyde generally formed as a consequence of lipid peroxidation (Turton et al., 1997). High MDA content correlates significantly with the amount of Zn in the intracellular space of the lichen (Cuny, 1999). There also occurs decrease in ATP content on transplants after exposure to pollutants such as heavy metals Pb, Cu, and Zn (Kardish et al., 1987; Garty et al., 1988). It is also seen that the lichens on exposure to mixed pollutant treatment result in swollen as well as degenerated chloroplast thylakoids (Tarhnen, 1998). It is also seen that the high heavy metal concentration results in higher ethylene production. Ethylene, a phytohormone a simple hydrocarbon gas and an endogenous regulator of plant growth seems to produce in small quantity in normal conditions but seems to increase during different stress factors such as; mechanical wounding, bruising, chilling, freezing, irradiation, attack by microorganism, heat salinity, drought and hypoxia (Liebermann, 1979) i.e., certain heavy metals such as Fe²⁺ act as a cofactor in the conversion of ACC to ethylene. Moreover, additional substances such as different cations, chelating agent scavengers of free radicals, inhibitors of phosphorylation and oxidative phosphorylation, considered to be effective in the transcription as well as translation of protein and potential precursors for ethylene. Thus, there occurs an increment in the amount of stress hormone ethylene on exposure of heavy metals (Schieleit & Ott, 1994). Vehicular and industrial contamination were considered as stress factors leading to an increased ethylene production (Epstein et al., 1986).

    METHODOLOGY

    Materials and Methods

    Lichen collection and transplantation

    The Lichen species taken for the study belongs to foliose growth form Pyxine cocoes (Physciaceae) (Fig. 2). The species was selected due to its high accumulative ability as well as its toxitolerant nature and frequent availability in the study area. The species was easily recognized due to its yellowish grey lobate thallus, found growing on tree trunks and twigs. The species is UV+ yellow as it contains lichexanthone and triterpenes.

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      The foliose growth form Pyxine cocoes

      Samples of Pyxine cocoes which belongs to the family Physciaceae collected from outskirts of the city, an unpolluted area of Aseni village of Barabanki district (Fig.3). The study area was situated between N26°53ˊ14.50ˊˊand E81°8ˊ46.10ˊˊ. The species was chosen due to its luxuriant growth in the tree barks of Mangifera indica as well as its tolerant ability to grow in stressed conditions and accumulative behavior of pollutants from the atmosphere. About 1–2 g of thallus was taken from the control site (Fig. 4). The sample which then collected are fixed in cardboard sheets using fevicol as well as cotton and are then transplanted in two study sites (Fig. 5). The study site 1, Rana Pratap Marg, chosen as a result of high vehicular pollution because of the main two-way road to link the city centre (Fig. 6). The second site chosen was the one-way road towards Sahara Ganj with less number of vehicular activity (Fig. 7). The samples are then fixed on the trees which are facing to the vehicular emission for 15 days of exposure.

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        Map of Barabanki district, the control site.

        IMG_20190705_130137_3.jpg
          .Collection of lichens from Control site – Barabanki district of Uttar Pradesh

          The IAP method, is based on the quality of the air in a determined area and hence evaluates the level of atmospheric pollution based on number, frequency and tolerance of the lichens present in the study area. About twenty different formulas are associated with IAP calculation method. The most frequently used method for IAP calculation is as following;

          IAP = Ʃ Fi

          where F, is the frequency of the ith species which is, calculated as no. of rectangles in the grid in which a given species occurs (Herzig & Urech, 1991).

          In the present study, the transplant method was adopted, to study the change in lichen material transplanted for a fortnight time period. The transplantation technique is performed in the areas where, lichen taxa exhibit their complete absence. This method enables the lichen to expose even in the lichen desert or areas where there is no substratum for lichen growth. The lichen thalli which are collected from the tree trucks (control) are fixed on suitable substratum using fevicol and cotton and are then exposed to monitoring areas to evaluate the health as well as the degree of the damage (Fig. 5). The number of vehicles, type of vehicles together with topography, road conditions also considered during the biomonitoring study in an area.

          IMG_20190708_154515.jpg
            Preparation of lichen transplants.

            Sample preparation for analysis

            Pigment analysis

            After 15 days of exposure to vehicular emission in two study sites, the samples were collected, and then scraped from the bark under a microscope using blade and distilled water. The scraped samples are then analyzed for Chl. a, Chl. b, total Chlorophyll content carotenoid as well as chlorophyll degradation. Replicates of 20 mg is weighed, to that added 10 ml 80 % acetone, and are crushed, then centrifuged for 15 min under 10,000 rpm and the supernatant is transferred to respective test tubes. The reading has been taken in 663, 645 nm, 510 and 480 nm respectively. The reading is noted and significant concentration is analyzed by Arnon’s method and carotenoid pigment calculated by Parson’s method.

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              Lichen transplant in Site 1 – Rana Pratap Marg (T1)

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                Lichen transplant in Site 2 - Sahara Ganj (T2)

                Chlorophyll degradation

                The remaining supernatant is collected in different test tube and kept overnight for analyzing chlorophyll degradation. After 24 hours, the reading for Chlorophyll degradation is taken at 435 and 415 nm respectively.

                Protein analysis

                Protein estimation was done by using Lowry’s method. The pellet of the sample was added in 1N NaOH kept overnight, warmed up at 55 °C for 10 minutes and were then centrifuged at 10,000 rpm for 15 minutes. The protein content was estimated using Folin’s phenol reagent with BSA as standard and readings were measured at 700 nm (Lowry et al., 1951).

                Heavy metal estimation

                The Heavy metal estimation was done by HPLC-ICP MS (7500cx). The replicates of 250 mg of sample was digested with HNO₃ as well as H₂O₂ and are then placed in hot plate until solvent gets evaporated and were filtered using Milli Q water and are ready for ICP-MS analysis. The readings are noted and calculations were done using respective formula.

                RESULT AND DISCUSSION

                Pigment Content

                The mean values of pigment content in the transplanted samples, illustrates that the Chl. A, total Chlorophyll contents were high in transplants than the control samples. The value ranged from 0.522±0.0521-0.875±0.11404 for Chl. A, and 0.7721±0.07622-1.5078±0.5178 for total Chl. whereas, Chl. B seems to lower in the transplanted samples than the control i.e., it ranged from 0.3110±0.2997-0.3279±0.0326. The result reveals that rainfall or heavy precipitation that especially occurred during transplantation in months of June-July dislodge the contaminants on the thallus surface. The involvement of heavy metals on the physiological effect seem to be less unaffected in transplanted samples.

                The pigment, carotenoid plays a critical role in the photosynthetic process, which showed an increasing trend as the stress level increases in lichen sample (Bajpai et al., 2011). The carotenoid concentration in transplants ranged from 0.3110±0.2997-0.3279±0.0326 thus, revealed stress confronted by the transplants on exposure to vehicular emissions.

                One of the most frequently used parameters in lichen stress physiology is chlorophyll degradation, which is expressed as phaeophytinization this, reflects the ratio of Chl. A to Phaeophytin A (Garty, 2001). Chlorophyll content and its degradation are often used as one of the easiest and more accurate method of biomonitoring, the ratio of the optical density of chlorophyll which read at 435 and 415 nm is mostly frequently used for the chlorophyll degradation. The chlorophyll degradation is more pronounced in transplants and it ranged from 0.968±0.00427-1.0568±0.049215. A ratio of 1.45 indicates that chlorophyll is unchanged and reduction in the value from 1.45 indicates that the thallus is under degradation (Garty et al., 2000). Figure 8 explains the gradation in the pigment content in controlled and transplanted samples.

                Protein Content

                The protein content is comparatively higher in the transplanted samples than the control. The first transplanted sample, from main two-way road to link the city Centre (Rana Pratap Marg) has the maximum protein concentration, the value ranges from 0.4875±0.31056-2.87718±0.8488 as shown in Figure 8. The probable reason for higher concentration of protein may be due to the stress resulted in production of HSPs for adaptation together with ethylene (stress hormone) (Schieleit & Ott, 1994). The protein concentration in the second transplanted site i.e., one-way road towards Sahara Ganj road seems to be less in amount compared with the control sample that is, ranged from 0.3297±0.285152-0.40588±0.218992 and 0.40044±0.2387-1.30818±0.8367 respectively. The declination in the protein content of transplanted sample from the second site may be due to the leach out of the heavy metal during precipitation which cause comparatively less pronounced stress in the lichen thallus.

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                  Graph showing the Pigments and Proteins concentration in Pyxine cocoes in controlled and transplanted sites.

                  Heavy Metal Content

                  A total of six metals Cd, Cr, Fe, Ni, Pb, and Zn were estimated and considered to be released in the atmosphere through vehicular activity. The transplanted experiment using lichens reveals that the heavy metal concentration is more in control than the transplants (Fig. 9). This helps to reveal the factors resulting in the leaching out of the heavy metals during accumulation mechanism. Metals can easily remove by rainfall or heavy precipitation, this dislodges the contaminants on the thallus surface (Brown & Brown, 1991). As a result, the indulgence of heavy metal on the physiological activities of lichen seems to be less, due to this leach out activity, chlorophyll degradation as well as protein content seems to be more pronounced in control samples than the transplanted one.

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                    Graph showing the variation of heavy metal accumulation in Pyxine cocoes in control and transplanted sites.

                    CONCLUSION

                    From the above observations, it is clear that the site having higher vehicular activities have higher concentration of heat shock proteins as compared to the second site, having low vehicular activity. A clearer picture of the accumulation pattern of heavy metal will be available if the experiment will be carried out during different seasons within a year.

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                    ACKNOWLEDGEMENTS

                    I express my gratitude and indebtedness to my project guide Dr. D.K. Upreti FNA, FNASc, CSIR- Emeritus Scientist for the memorable guidance, facilities extended and constant encouragement enabling me to fulfil my dream of completing the present work. I had unique opportunity for the discussion of my topic with Dr. Sanjeeva Nayaka, Senior Principal Scientist, CSIR-NBRI. My heartfelt gratitude to Dr. Siljo Joseph, INSA-DST INSPIRE Faculty, CSIR-NBRI for his consistent encouragement and support. I will be failing in my duty if I don’t thank Ms. Kirti Kumari, PhD scholar who helped me all through my research. I am thankful to Dr. Rajesh Bajpai for his help in arranging literature and guidance. I express my wholehearted gratitude to Dr. G.K. Mishra and Mr. Komal Kumar Ingle who guided me in the field work and identification of lichen samples. I also thank Dr. Geetgovind Sinam for his technical assistance and generosity. It is a pleasure to acknowledge all the research scholars of lichenology laboratory, CSIR-NBRI for the bounteous help and support especially Mr. Roshinikumar Ngangom and Ms. Jyotsna Chakarworti. I also extend my gratitude to Mr. Akhil C A, Mr. Ranjith Layola M R, and Mr. Abhinav Sharma for their consistent help during my study.

                       

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