Diversity and distribution of corticolous lichens and their relationship to levels of nitrogen dioxide in chiang mai province thailand

BALIDO DIVERSITY AND DISTRIBUTION OF CORTICOLOUS LICHENS AND THEIR RELATIONSHIP TO LEVELS OF NITROGEN DIOXIDE IN CHIANG MAI PROVINCE, THAILAND BACHELOR THESIS Study Mode: Full-time

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THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

FRANCINA MAE A BALIDO

DIVERSITY AND DISTRIBUTION OF CORTICOLOUS LICHENS AND THEIR RELATIONSHIP TO LEVELS OF NITROGEN DIOXIDE IN

CHIANG MAI PROVINCE, THAILAND

BACHELOR THESIS

Study Mode: Full-time

Major: Environmental Science and Management

Faculty: Advanced Education Program

Batch: K46

Thai Nguyen, 15/11/2018

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Environmental Science and Management

Student Name Francina Mae A Balido

Student ID DTN1434290109

Thesis Title Diversity and Distribution of Corticolous Lichens

And Their Relationship to Levels of Nitrogen Dioxide

in Chiang Mai Province, ThailandSupervisor(s) 1 Asst Prof Dr Wanaruk Saipunkaew (Thailand)

2 Dr Ho Ngoc Son (Vietnam) Supervisor’s

signatures

Abstract

The study aimed to investigate the diversity and distribution of lichens and

their relationship to levels of nitrogen dioxide within the basin of Chiang Mai Province The study evaluated six districts which were Mae Tang, Mae Rim, Doi

Saket, Mae On, San Pa Tong and Chiang Mai City 10 mango trees (Mangifera indica

L.) were selected for lichen investigation in sites A total of 14 families, 22 genera

and 38 species were found Thirty- eight species consists of 24 crustose lichens and

14 foliose lichens The richness and diversity of lichens were determined by using Shannon Diversity Index The highest diversity index was found in Mae Tang and the lowest diversity index recorded was in Chiang Mai City The highest richness was both discovered in Mae Tang and Mae On and the lowest richness was found in Doi Saket Bray-Curtis Similarity Index was used to determine the similarity of lichens communities and the distribution of lichens among sites was illustrated by using Detrended Correspondence Analysis (DCA) The results indicated two main groups within the six study sites and 30 % similarity were found between the groups

Lichens species such as Pyxine cocoes, Hyperphyscia adglutinata, Phyllopeltula cf

corticola, Rinodina roboris and Dirinaria picta were found in urban and suburban

areas Pyxine cocoes, Hyperphyscia adglutinata and Phyllopeltula cf corticola were identified as tolerant species to air pollution Dirinaria picta was identified as

sensitive species on air pollution The lowest NO2 concentration (9.5 ppbv) was distinguished in Mae On and highest concentrations (19.1 ppbv) were found in Mae Tang, Doi Saket, San Pa Tong and Chiang Mai City Pearson’s correlation coefficient was used to correlate all factors and lichen diversity, whereas the results showed that

there is no significant correlation between nitrogen dioxide, environmental factors, species richness and lichen diversity in the study

Keywords: lichen diversity, lichen distribution, NO2 concentrations, passive

sampler

Number of pages 67 pages

Date of

submission

15/11/2018

ACKNOWLEDGEMENT

My sincerest thanks to my thesis supervisors, Dr Wanaruk Saipunkaew and

Dr Ho Ngoc Son for the kind supervision and guidance which both helped me to

do my thesis at my best In particular, I want to give thanks to Dr Wanaruk Saipunkaew for giving me opportunity to worked on my thesis topic “DIVERSITY AND DISTRIBUTION OF CORTICOLOUS LICHENS AND THEIR RELATIONSHIP TO LEVELS OF NITROGEN DIOXIDE IN CHIANG MAI PROVINCE “that I can never finished without the help of my laboratory members which were MS Student, Sujinda Bungwan, Ph D students, Nattakarn Sransupphasirigul and Chitsupang, Kheawsalab Jiraporn Saenthiya for the help in collecting, and identifying lichen specimens My appreciation also extends for,

MS Student View Varaphan for assistance in using pH meter and for Dr Sompron Chantara for approval of using passive samplers on her laboratory and for the assistance of her laboratory students that were MS Student Sukanya Prawan, MS Student Patcharee Saejiw and MS Student Tantaraporn Charoen in preparing and using the passive samplers I also want to acknowledge the Head of the Faculty of Science, Dr Chitchol Phalaraksh for the appreciation of our work in our research

in Chiang Mai University I want express my gratitude to Thai Nguyen University, Chiang Mai University, ASEAN International Mobility of Student (AIMS) for the opportunity and support for their students on this internship I would like to express

my gratitude Dr Ho Ngoc Son for choosing me as one of the students he would like to handle And I want specifically to convey my thanks for his motivation and

encouragement to do well even in making my bachelor thesis even before and after the conduction of data

My sincerest gratitude to my family and relatives for the endless and genuine support and understanding on my decisions within the span of living abroad for studying and making thesis For that, I become more confident and matured than before The blessings and challenges I faced were all worth it with these people all behind my back and will strive to do better in the future I also want to acknowledge my school “Refiners Christian School” which I studied for 9 years and gave me the chance to study abroad which was I am not expecting to do

so and to our Administrator,Mr Marjun Macaleng, Mrs Irene Macaleng, School Principal Mrs Cherry Tanyag and Ms Elsa Belen for their encouragement for us students to venture to this opportunity and be equipped students I want to convey

my gratitude to my longtime friends which are both in the Philippines and close friends studying with me which were in Thai Nguyen University for being there and being each other support and strength whenever I am in good situation and in

a bad situation I want to specially express my thanks to Kristina Reyes, my roommate which is a close friend that is very helpful and gave advices whenever I had worries and Veatriz Malitic for the understanding me and for sharing same interests with me especially in music Joy Ongkiatco for being such a honest and caring friend to me, Enzo Bernardo and Luis Chavez for being such good friend to

me since before until now that proves whenever we understand each other in worrying times Jessica Cosico, Aj De Castro, Lester Lagansua, Ghia Sarmiento and King Reyes which were comfortable friends that I can joke and play with and

same goes for the laughter you had brought to me and to other as well My Ate’s and Kuya’s from other batch for looking after me and us younger Filipinos and gave advices whenever it is about academics and in life situations

My Vietnamese friends which were I am thankful were Chi Ha Trang, Chi Qunyh Trang, Chi Luyen Nguyen, Chi Minchau Nguyen Chi Duong Hoàng and all the Chi and Anh in K46B class who were like my older sisters and brothers that treats me well My special thanks also to Pi Puangpech Pongpa for the friendliness and generosity of your time for me and all of us who are new in Thailand The memories of my college journey were about to end and will be forever cherished, completed and remembered with the friendship that felt already like a family whenever and I hope to continue for a very long time.I also want to congratulate myself for holding and giving all the best You’ve worked hard And save the best for last, I want to thank God for everything, the guidance, blessings and challenges which helped me to grow You showed me what will happen if I will just follow you and by that, I entrust and give back all the achievements and experiences I’ve earned for your own glory

Student

Balido, Francina Mae A

TABLE OF CONTENTS

LIST OF FIGURES viii

LIST OF TABLES ix

LIST OF ABBREVIATIONS x

PART I INTRODUCTION 1

1.1 Research rationale 1

1.2 Research objectives 3

1.3 Research Questions and Hypotheses 3

1.3.1 Research Questions 3

1.3.2 Research Hypothesis 4

1.4 Limitations 4

1.5 Definitions 4

PART II LITERATURE REVIEW 5

2.1 Lichens 5

2.2 Morphology 5

2.3 Growth forms and reproduction 7

2.4 Factors affecting the lichens 9

2.5 Lichens as biomonitors of air pollution 10

2.6 Passive Sampling 13

PART III MATERIALS AND METHODS 17

3.1 Time frame and description of the study areas 17

3.2 Materials 20

3.3 Methods 21

3.3.1 Lichens Study 21

3.3.2 Passive Sampling 23

a) Preparation of the solutions 23

b) Preparation of diffusion tube 23

c) Exposure of the diffusion tube 24

d) Extraction of the sample 25

e) Analysis of the sample 25

3.3.3 Data Analysis 26

a) Lichens Diversity: Shannon Index 26

b) Similarity of lichen communities and distribution of lichens 27

c) Correlation between nitrogen dioxide, environmental factors,

PART IV RESULTS AND DISCUSSION 29

4.1 Results and Discussion 29

4.1.1 Lichens Diversity 29

4.1.2 Similarity of lichen communities and distribution of lichens 40

4.1.3 Environmental factors in the study sites 44

4.1.4 NO2 concentration in the study sites 45

4.1.5 Correlation between nitrogen dioxide, environmental factors, species richness and lichens diversity 46

PART V CONCLUSION AND RECOMMENDATION 49

5.1 Conclusion 49

5.2 Recommendation 51

REFERENCES 52

APPENDICES 60

LIST OF FIGURES

Figure 1 Three types of lichens (a) crustose (b) foliose (c) fruticose………….8

Figure 2 The process of gas diffusion in the passive sampling procedure…….16

Figure 3 Location of the six selected areas within Chiang Mai Province 19

Figure 4 The position of a grid frame on selected tree 22

Figure 5 Configuration of passive sampler……… ……… 24

Figure 6 Analysis of the sample using NO2 test kit……….25

Figure 7 The NO2 standard color chart……… ……… ………… 26

Figure 8 Dendrogram of similarity of lichen communities……….…42

Figure 9 Distribution of lichens in all study sites……… 43

LIST OF TABLES

Table 1 Location of the six study sites Site number (No.), Study sites, longitude

and latitude and elevation……….………… 18

Table 2 List of lichens families, genera and species of lichens found in the study areas……… 30

Table 3 List of lichen species and their frequencies in all study areas 34

Table 4 List of richness and diversity index in the study areas………… 39

Table 5 List of environmental factors collected in study sites………… 44

Table 6 List of the NO2 concentrations in study sites……… 45

Table 7 Pearson correlation coefficient between (r) eight parameters…… 48

LIST OF ABBREVIATIONS DCA Detrended correspondence analysis

GF/A Glass Microfiber filters

GPS Global Positioning System

MVSP Multivariate Package Program

NO 2 Nitrogen dioxide

PM Particulate matter

ppbv parts per billion by volume

SO 2 Sulphur dioxide

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PART I INTRODUCTION 1.1 Research rationale

The economic growth of Thailand causes several issues which has come to

a point where it is negatively affecting its people and environment Air pollution

is one of the issues that beset the country as it is significantly worsening problem

in recent times Several health implications concerning on respiratory- related conditions increased and caused 50, 000 deaths per year (Pacific Prime Thailand, 2017) In which particularly, Chiang Mai Province located in Northern Thailand greatly showed rapid growth of its economy since the number of tourist attractions, accommodations, factories, restaurants, road constructions and business buildings have increased as well as the number of tourists visited Chiang Mai Province Moreover, the industry and service sectors were considered to be the top two largest laborers in the province over the past few years (Kitirianglarp, 2015)

By these gains of the economic sector, there were trade-offs observed in the province, these are the traffic congestion, poor waste management, water and air pollution which generally affect the natural environment and resources of the province And out of these problems, air pollution is the biggest and greatly affects most of the people in terms of health According to the Pollution Control Department, PM 2.5 levels recorded on March 18, 2018 ranged from 48 – 86 micrograms per cubic meter of air which is high compared on the safety level of

50 micrograms per cubic meter of air

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Annually, there are many cases wherein the residents and tourists in the hospital complain some illnesses and even caused several deaths because of the pollution These deaths caused by the excessive exposure to hazardous pollutants such as sulphur dioxide, benzene, lead, carbon monoxide and nitrogen oxide Particularly, sulphur dioxide causes shortness of breath, benzene can cause leukemia (ATDSR, 2007) and lead affects the central nervous system and the brain (WHO, 2018) On the other hand, too much exposure to carbon monoxide can lead

to death (NCEH, 2017) and respiratory problems when someone intake nitrogen oxide in extremely high amount (US EPA, 2016)

In general, conception of imposing standard levels of nitrogen dioxide aimed to protect human health Pollution Control Department under the Ministry

of Natural Sources and Management in Thailand set the standard levels for the hazardous pollutants in Thailand to compare to the present levels in an hour, month and a year

Likewise, other ways of monitoring the air pollution is considered to be useful and helpful These ways, for example is using lichens as biomonitoring method (Blasco et al., 2008) and by the use passive sampler which collects the data

of nitrogen dioxide levels which is physicochemical monitoring method Both methods are known and commonly used in determining air pollution as the biomonitoring method determine the effects of the pollution to organisms and the physicochemical method identifies the concentration of the hazardous pollutants

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1.2 Research objectives

With that, the objectives of the study are to investigate lichens diversity and their distribution of the lichens within the basin of Chiang Mai Province, to monitor the environmental factors and nitrogen dioxide levels and to determine the correlation between nitrogen dioxide, environmental factors, species richness and lichen diversity

1.3 Research Questions and Hypotheses

1.3.1 Research Questions

The research questions are based on the objectives of the study which are

the following:

1 What is lichen diversity in Chiang Mai Province?

2 What is the similarity of the species communities and lichen distribution

within the study sites?

3 What is the light intensity, temperature, relative humidity, bark pH,

elevation, species richness and nitrogen dioxide levels in each site?

4 What is the correlation between nitrogen dioxide levels, environmental

factors (light intensity, temperature, relative humidity, elevation and bark pH), species richness and lichen diversity in Chiang Mai Province?

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1.3.2 Research Hypothesis

The study comprises two hypotheses such as

1 Null Hypotheses: No significant correlation between nitrogen dioxide levels, environmental factors, species richness and lichen diversity

2 Alternative Hypotheses: Significant correlation between nitrogen dioxide

levels, environmental factors, species richness and lichen diversity

1.4 Limitations

The limitation of the study was the lack amount of time in conducting the research which results on a short observation on lichens diversity and distribution Usage of only capable equipment on producing results within short study period was considered such as the passive sampler Passive sampler is known as a simple and easy device on collecting nitrogen dioxide levels

The mycobiont components in lichens are from the two groups of fungi which are Ascomycetes (common) and Basidiomycetes (rare) group whereas, the common photobionts for phycobiont are Trentepohlia and Trebouxia and for

cyanobiont are Stigonema, Nostoc, and Scytonema (Wolseley et al., 2015) Also,

it is a composite organism that is found in many substrates such as soil (terricolous lichens), bark (corticolous lichens), rocks (saxicolous lichens) and habitats (mountains, seashores and rainforests) which contributes in some important process such as the process of slowing down the soil formation and providing food and shelter for animals

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2.2 Morphology

The structure of the lichens is comprised of its body which is made up of filaments of fungal cells and this lichen body is called thallus (singular thalli) It has two types based on the internal structure namely homoiomerous thalli that is a simple type of thallus having only the filaments of fungal cells and algae in it while heteromerous thalli is a layered type wherein it is composed of four parts These parts are the upper cortex, algae layer, medulla and lower cortex (Constantine et

al., 2018)

The upper cortex is the outer and protective layer covering of the thallus while the algae layer is the photosynthetic area in the thallus and whereas the algae associates with fungal hyphae For the medulla, it is the central foundation of the whole structure of thallus having firm walls and capable of acquiring gases in lichens The fungal hyphae in the medulla layer is uncompact but on the lower cortex, it has compacted fungal hyphae (Lepp, 2011b) And whereas some hyphae from the bottom termed as rhizines serve as roots to stick into subtrates is present and when the lower cortex is absent in the structure, the hypothallus is formed from

a small sheet of hyphae to do the role

Alongside with that, there are other structures such as hapter, holdfast cephalodia, cilia, pruina, cyphellae and pseudocyphellae in lichens Hapter and holdfast help lichens attach on subtrates besides rhizines Cephalodia are small

structures found only when lichen has both cyanobacteria and green algae (Henskens et al., 2012) These small structures usually in the upper or lower part

2.3 Growth forms and reproduction

Lichens have three common growth forms which are crustose, foliose and fruticose (Figure 1) Crustose lichen is the most common growth form and this type of lichen is a crust-like lichen and firmly attached to its substrates while foliose lichen is the type of lichens that has flattened shapes It is also known as leaf-like lichen that loosely binds on its substrate Fruticose lichen shown in Figure

1 (Sharnoff, 2018) is a shrub like or hair like lichens that grows in substrate by only attachment of its lower part since it obtains multidimensional forms (Lepp, 2011b) The growth of these crustose, foliose and fruticose lichens per year is usually around 0.5-5 mm

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(b) (c)

Reproduction of lichens is done by sexual and asexual (vegetative) reproduction In sexual reproduction, only the fungal component is being reproduced and there are fruiting bodies that have structures which are commonly known as ascus and basidium Ascus and basidium structures are particularly like container of spores that are being produced by two fungal partners such as Ascomycetes and Basiodiomycetes Ascomycetes produce spores called ascopores and Basiodiomycetes carried out spores named as basidiospores

In addition, these fruiting bodies are distinguished by their different forms namely apothecia and perithecia Apothecia are cup-like fruiting bodies found in the upper surface and can be differentiated into three kinds: lecanorine, lecideine, and lirellae For lecanorine apothecia, it has algae and margin having the same color with its thallus However, there is no margin, algae and have different color when it is a lecideine apothecia(Smith, 1921).Lirellae apothecia is long in shape and specifically attain black colored margins On the other hand, perithecia are spherical-like fruiting bodies whereas it only has small opening unlike apothecia

Figure 1 Three types of lichens (a) crustose (b) foliose (c) fruticose

(a)

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Asexual or vegetative reproduction for lichens involves three structures These include isidia, soredia and lobules and unlike from the sexual reproduction both the algal and fungal components are reproduced Isidia are barrel-shaped or coral-like structure, soredia are small and powdery group of hyphae and lobules are flat lobes in the thallus These structures detached on the thallus and reproduce

by the environmental conditions and physical interactions, for example by wind

and disturbance of animals (Smith, 1921)

2.4 Factors affecting the lichens

Lichens are composite organisms found on variety of habitats where there are substrate and environmental conditions for their growth The substrate conditions, for instance are surface types and components Lichens are generally having high frequency on rough, flaky and persistence barks and low frequency on smooth barks Likewise, for the surface components including the pH value and water content on the tree barks, lichens can survive even though there is little amount of water available and has moderate amount of acidity in the substrates (Lam et al., 2013)

Environmental conditions affecting the lichens were temperature, relative humidity, light intensity and elevation Both environments that have high and low temperature alter the richness of lichens because the reproduction and the food of lichens are limited by these temperatures The factor of humidity in general, influences the lichen growth at which point is more evident in high humid locations

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than the places that have low humidity (Čabrajić, 2009).The light intensity upon lichens is also one of environmental conditions considered as the sunlight plays an important role for lichens to exist And for that, lichens are present in places that

have enough exposure on sunlight (Lam et al., 2013) The growth and diversity of

lichens is also influenced by elevation, as higher elevation the more diverse and species of lichens were observed (Kumar et al., 2014)

2.5 Lichens as biomonitor of air pollution

Both sensitivity and adaptive capability of the lichens is considered to be essential in monitoring environmental problems particularly the air pollution The sensitivity of lichens is caused by their structure which has the absence of an outer layer like epidermis or cuticle and roots In addition to that, the adaptive capability pertains on the structural variations of lichens depending on the environment it grows With that, lichens are exposed and notably influenced by their atmospheric conditions ascertaining their relevancy as an indicator of air pollution (Lawrey, 2018)

Crustose, foliose and fruticose lichens have their own level of tolerance on the changes in the environment Within these lichens, crustose lichens are known

to be the most tolerant lichen species since it is still present even in areas that have polluted air Whereas, foliose lichens can only grow in partly polluted areas and fruticose lichens cannot live totally on places that has really small amount of pollutants since it requires completely clean air for it to grow (BBC, 2014) Likewise, the determination of sensitive (absent) and tolerant (present) species

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within the three common lichens specifically helps in distinguishing the possible indicator species as well as the changes in the environment

With that, some studies evaluated the global environmental problem which

is climate change by using the responses of lichens in terms of the diversity and abundance within the areas of study As for example, a sixteen –year (1993- 2009) study in US Pacific Northwest determined that the high elevation lichens were the most affected since the number of species and the distribution decreased comparing on the low elevation concluding that the warming greatly affect the cooler environments (Glavich et al., 2018) A study from North-western Alaska observed low number of lichens in the Arctic tundra (Joly et al., 2009)

And, a study on cold temperature species of lichens decline in both highland specifically in the mountains of Western and Central Europe and lowland areas (Shukla et al., 2018) Nevertheless, from some studies lichens were increasing in cold climates rather than decreasing A study on forests in Sweden whereas

specific species were investigated lichens such as Bryoria spp., Usnea spp and

Alectoria sarmentosa (Ach.) Ach The Usnea spp and Alectoria sarmentosa

(Ach.) Ach appeared to increase in the areas that have cold temperature than in warm temperature places while the Bryoria spp was not present all in both

temperatures (Esseen et al., 2016) Moreover, climate change increased of either

the extreme heat or rainfall, the growth and the diversity of lichens will still be affected

Therefore, lichens in local climate scale were considered to be influenced

by the varying levels of microclimatic factors from each environment it grows In

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a study situated in Badrinath valley in Western Himalaya, the lichens were identified to be affected by the temperature, altitude and relative humidity Similarly, distribution study of lichens was altered in the White Mountains of California because of the light intensity (Gupta et al, 2014)

And in accordance of the sensitivity and tolerance capability of lichens, these were used to assess a common environmental problem which is air pollution Air pollution pertains on the harmful and excessive amount of pollutants released

in the air caused by the intensified human activities For instance, these activities were vehicle emissions, power plants, agricultural and industrial activities and from that, the common pollutants released are sulphur dioxide and nitrogen dioxide

With that, the possible availability and excessive amount of atmospheric pollutants such as nitrogen dioxide and sulphur dioxide were monitored by using lichens (Richardson ,1988) As both high amount of nitrogen dioxide and sulphur dioxide negatively affect the chlorophyll and eventually kills the lichen Due to that, studies about both atmospheric pollutant levels used particularly the sensitive and tolerant species of lichens on each pollutant

Based on that, there was a study that discovered new species of lichens and occurrence again of some species in London conforming the air quality became free from sulphur dioxide (Hawksworth & McManus, 1989) In United Kingdom, the nitrogen dioxide pollution was observed as five terricolous alpine lichen species were affected which were sensitive lichen species to nitrogen dioxide (Nash & Gries, 2002)

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A study in Colombo and suburbs that determined the amount of sulphur dioxide and nitrogen dioxide pollution evaluated lichens as well as the disturbances found in the area of the study The pollution within the area was confirmed to be in high amount since there was a declined in number of lichens as well as the total absence of tropical lichens Considering that, these tropical lichens were generally known as pollution sensitive species in the study in area of Colombo and suburbs (Attanayaka et al 2007)

Muhammad et al (2018) identified the D picta species of lichens as the

species associated with the vehicular emissions in Malaysia particularly in Bahut

Pahut area Since, D picta was present in all locations whether it has high traffic

or low traffic level and considered to be the tolerant lichen species among all the present lichens Alongside with that, a study in Chiang Mai, Thailand determined

that P cocoes was tolerant to air pollution And also determined the sensitivity of

D picta which may be considered as a bioindicator of air pollution within the area

and the tolerance of P.cocoes (Pimwong, 2002)

2.6 Passive Sampling

The device used in this method is called passive sampler wherein it performs molecular diffusion to collect the pollutants In this procedure, there is a free flow of analytes (heavy metals, inorganic and organic compounds) from the atmosphere to collecting media illustrated in Figure 2 (sigmaaldrich, 2010) These compounds can be in any matrices such as soil, water and air (Wongniramaikul, 2012) Furthermore, the average amount from the collected pollutants were estimated by using the Fick’s law of diffusion

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Passive Sampling is a physico- chemical method commonly used in air pollution monitoring This method is also considered to be cost- effective and flexible tool in assessing the amount of pollutants in any study area It is a cost- effective device because it does not require any maintenance for it to work properly and electricity to operate as the same time as it is a flexible tool by being a lightweight and simple device

Due to that, some studies emerged using passive sampling methods in their respective countries with the purpose of monitoring air pollutants and their amount which affects the air quality in the country In Malaysia, the results from the passive sampling proved the impact of the human activities, specifically the construction site and vehicle emissions on the air quality in the area Considering, three areas were chosen and among these, the farthest from the roads and the construction site showed a 14.0 ppbv which was the lowest amount while the area that was near to these activities displayed a 23.4 ppbv of nitrogen dioxide amount and the highest nitrogen dioxide amount recorded was 36 8 ppbv which were measured in the areas that was nearest to the activities (Chong et al., 2017) A monitoring study in the major highways in Abeokuta, Nigeria determined air pollutants like nitrogen dioxide, sulphur dioxide, carbon dioxide and other pollutants in both wet and dry seasons And all the pollutants appeared to be in high concentration in the dry season than wet season For instance, the Iyana Mortuary have the highest concentration of both nitrogen dioxide and sulphur dioxide This can be associated with thedomestic cooking, expanding of roads and traffic congestion Likewise, the high concentration of carbon dioxide in Kuto was

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because of the vehicular emissions and particularly the concentration of this pollutant was higher than the WHO (2005) threshold limit value (TLV) of 500 ppm indicating the air quality in the area was not in a good condition anymore and can

severely affect the health of the people (Olaynika et al., 2015)

Another study in Chiang Mai Thailand wherein the NO2 concentrations within the urban, suburban and rural areas in Chiang Mai, Thailand were investigated and determined the high concentration in urban areas compared to the suburban and rural areas Particularly, the area that displayed the highest concentration was evidently located near where there was a common and serious traffic problem

Some studies that both used passive sampling and lichens in evaluating the air quality were found A study in South Korea evaluated the sulphur dioxide, nitrogen dioxide in different types of forest (Quercus, coniferous and deciduous) The deciduous forest was located in the Korea Peninsula which was more prone

on the impact of human activities as coniferous forest and the Quescus forest was located in Jeju island that obtain high relative humidity level Due to that, coniferous forest had the highest amount of nitrogen dioxide while deciduous forest had the highest concentration of sulphur dioxide and the Quercus forest had the lowest amount for both pollutants The results of the study showed a negative correlation of SO2 and NO2 concentrations to the diversity of lichens observed in the sites (Udeni et al., 2016) A study of Pomphueak (2005) in Chiang Mai, Thailand showed a positive correlation of the lichen diversity with theSO2 and

NO2 concentrations collected by passive sampling method

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Figure 2 The process of gas diffusion in the passive sampling procedure

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PART III MATERIALS AND METHODS

3.1 Time frame and description of the study areas

Lichens were collected over the period of April 27 to May 18, 2018 within the six study areas in Chiang Mai Province

Chiang Mai is a province in Northern Thailand and considered as the second – largest province in the country The province covers about 20,170.1057 sq (Chiang Mai Province official site, 2009) and the annual temperature is 25.6°C and

annual rainfall in the province is 1184 mm(Climate-Data.Org, 2017) The climatic

condition within the province is affected by tropical monsoons whereas three

seasons were observed such as cold season that begins during November and

February and the hot season during March – May Wet season begins in the month

of May- October (World Weather and Climate Information, 2016)

In particular, the six study areas were selected The study areas investigated are Mae Tang, Mae Rim, Doi Saket, Mae On, San Pa Tong, and Chiang Mai City

In particular,these areas chosen for the study are based on the degree of human

activities present within these places

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Table 1 Location of the six study sites Site number (No.), Study sites,

longitude and latitude and elevation

Elevation (mASL)

1 Mae Rim MR 18°54'50"N 98°56'42" E 320

2 Mae Tang MT 19°7'19" N 98°56'37"E 330

3 Doi Saket DSK 18°52'13"N 99°8'12" E 320

4 Mae On MO 18°46'35" N 99°14' 53"E 390

5 San Pa Tong SPT 18°37' 43" N 98° 53'44"E 300

6 Chiang Mai City CM city 18°47'43"N 98° 59' 55"E 310

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Figure 3 Location of the six selected areas within Chiang Mai Province

(Source: Tourism thailand, 2011)

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3.2 Materials

The following materials were used in collecting and analyzing the lichens and nitrogen dioxide (passive sampling method) within the study Materials used for collecting data for lichens were compass, pens, recording forms, pocketknife, grid frame, basket, camera, small paper envelopes, dropper, measuring tape and for nitrogen dioxide were cool box and plastic zip lock bag The laboratory materials for lichens study were the following: slide and cover glass, hand lens, 25-600 ml beaker, 10 ml graduated cylinder, analytical balance, pH meter, compound microscope, stereo microscope, ultraviolet lamp, GPS (Global Positioning System), Lux meter and wet and dry thermometer For the passive sampling method, the materials were oven, ultrasonic bath, shelter, 10 ml polypropylene tubes, parafilm, Whatman (GF/A), 10 ml syringe, 50 ml beaker, 10

ml micropipettes, 25 - 250 ml volumetric flask, glass stirring rods and pipet bulb

The chemicals used for lichens study were deionized water, sodium hypochlorite, potassium hydroxide; K :10%; Lugol’s iodine: 5% For the passive sampling method, the chemicals used were the following: deionized water, phosphoric acid, Triethanolamine (TEA), N-(1- Napthyl) ethylenediamine dihydrochloride (NEDA) and Sulfanilamide; C6H8N2O2S

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3.3 Methods

3.3.1 Lichens Study

a) Lichen collection

The study investigated 10 mango trees (Mangifera indica L.) trees within

an area size of 2 km x 2 km Grid frame was used to count the frequency of lichens

on the selected tree The grid frame size was about 20 x 50 cm2 and had 10 subsquares, size of 10 x 10 cm2 It is attached on the part of the tree trunk where covered with lichen most has many lichens and placed 150 m above ground by using a rope (Figure 4) Species and frequency of lichens found inside the grid frame were collected and listed in the recording forms by using hand lens To avoid misidentification of lichens within the grid frame, lichens that had a smaller diameter than 3 mm was not collected Whereas, lichens outside the frame were also determined and its frequency was given as one

The lichens that were not recognized in the field was collected in paper envelopes for further identification by using ultraviolet lamp, compound microscope and stereo microscope Morphology, growth, reproduction and dispersal of these lichens were identified by using key of Sipman (2003), key of Wolseley and Aquire- Hudson (1997) and key of Awasthi (1991) The circumference of the tree, type of bark, bark pH, aspects whereas the grid frame were placed in the tree trunk, degree of exposure of the tree in the sun and the description of the location, possible activities and establishments near the area whereas the distance of the trees was also determined in each site

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b) Measurement of some environmental factors

Some factors were collected by using equipment such as Lux meter for light intensity, Global Positioning System (GPS) for altitude, dry thermometer for temperature and wet - dry thermometer for determination of the relative humidity

by being hung at 1.5 meters above the ground The elevation from each site was adapted by using Google Earth

c) Analysis of bark pH

The collection of barks without lichens was conducted at 1 meter above the ground These barks are collected inside the paper envelopes and stored inside the freezer After that, the barks were weighted in the analytical balance having 0.5 grams and be kept in the plastic cups Then, they are soaked with 5 ml of distilled water for 8 hours Electrochemical analyser for pH meter was used to measure the bark pH from each 10 trees in six sites.

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3.3.2 Passive Sampling

The sites were chosen from one of the 10 investigated trees from each site The safety and required exposure of the passive sampler was also considered to avoid cases of lost passive samplers within the study The following procedures were the following:

a) Preparation of the solutions

The 20 ml of Triethanolamine solution was adjusted to volume with deionized water in 100 ml volumetric flask by using micropipette and a pipet bulb Saltzman reagent was prepared by mixing the reagent A and B The reagent A was made up of 5.375 g of Sulfanilamide solution and 14 ml of phosphoric which was adjusted to volume with deionized water in a 250 ml beaker

The reagent B was 0.038 g of N-( 1- Naphthyl) ethylenediamine dihydrochloride was adjusted to volume by using 25 ml beaker The Saltzman reagent was placed on the freezer until the collection day

b) Preparation of diffusion tube

The GF/A filter paper having a 110 diameter was cut into small pieces to fit

in the polypropylene tubes then were soaked in the deionized water for three times and put in the oven to prevent it to be stretched and for it to be dry

Forty-eight polypropylene tubes were prepared and placed on six passive samplers having eight tubes each These eight polypropylene tubes were divided into 5 diffusion tubes represented as replication of the samples and 3 blank tubes served as the control samples in same condition (Figure 5) 20% of

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Triethanolamine mixed with deionized water was added on the filter paper placed

in the bottom of all tubes

c) Exposure of the diffusion tube

The tubes were positioned vertically in the passive sampler and was placed

at 150 meters above the ground in one of the trees in each site The exposure period was during May 25 to June 1, 2018

On the collection of the passive samplers, the tubes were sealed with paraffin film and placed in plastic zip lock bag The tubes were kept in freezer until the extraction of the samples

Figure 5 Configuration of passive sampler

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d) Extraction of the sample

The extraction of the samples was done by adding 2 ml deionized water in the tubes using a syringe The tubes were being shaken for about 10 minutes before analysis

e) Analysis of the sample

The samples were transferred to small bottles and added 2 ml Saltzman reagent (Figure 6) The colors of the samples were compared to the NO2 standard color chart and determined the rates of NO2 levels (Figure 7)

Figure 6 Analysis of the sample using NO 2 test kit

a) Lichens Diversity: Shannon Index

Since the focus of the study is lichen diversity, the diversity index that is used for analyzing the data is Shannon Diversity Index The Shannon Diversity Index (H') is another commonly used index that is used to illustrate the species diversity in a community as it accounts for both the abundance and the evenness

of the species present

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The resulting product is summed across species and multiplied by -1 Below

is the following formula (Kerkhoff, 2010);

Η′ = − ∑(𝑃𝑖 𝐼𝑛𝑃𝑖)

H' = Shannon diversity index

𝑃𝑖 = proportion of the individuals found in species i

𝑛𝑖 = the number of individuals in species i

N = total number of individuals in a community

∑ = sum of the calculations

ln= natural logarithm

b) Similarity of lichen communities and distribution of lichens

The similarity of the lichen communities within all the sites was done by using cluster analysis andanalyzed by Multivariate Package Program (MVSP) The MVSP program used Bray -Curtis Similarity Index and illustrated in a dendrogram Detrended correspondence analysis (DCA) illustrated the distribution

of lichens in the study by using Past3 software