Source identification of polycyclic aromatic hydrocarbons in terrestrial soils in Chile

Journal Pre-proof Source Identification of Polycyclic Aromatic Hydrocarbons in Terrestrial Soils in Chile Woranuch Deelaman, Siwatt Pongpiachan, Danai Tipmanee, Chomsri Choochuay, Natthapong Iadtem, Oramas Suttinun, Qiyuan Wang, Li Xing, Guohui Li, Yongming Han, Muhammad Zaffar Hashmi, Junji Cao PII:

S0895-9811(20)30027-4

DOI:

https://doi.org/10.1016/j.jsames.2020.102514

Reference:

SAMES 102514

To appear in:

Journal of South American Earth Sciences

Received Date: 7 December 2019 Revised Date:

28 January 2020

Accepted Date: 29 January 2020

Please cite this article as: Deelaman, W., Pongpiachan, S., Tipmanee, D., Choochuay, C., Iadtem, N., Suttinun, O., Wang, Q., Xing, L., Li, G., Han, Y., Hashmi, M.Z., Cao, J., Source Identification of Polycyclic Aromatic Hydrocarbons in Terrestrial Soils in Chile, Journal of South American Earth Sciences, https://doi.org/10.1016/j.jsames.2020.102514. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.

Source Identification of Polycyclic Aromatic Hydrocarbons in Terrestrial Soils in Chile

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Woranuch Deelamana, Siwatt Pongpiachanb*, Danai Tipmaneec, Chomsri Choochuaya, Natthapong Iadtema, Oramas Suttinuna, Qiyuan Wangd, Li Xingd, Guohui Lid, Yongming Hand, Muhammad Zaffar Hashmie, Junji Caod

4 5 6 7 8 9 10

a

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b

Faculty of Environmental Management, Prince of Songkla University Hat-Yai Campus, Songkla, 90112, THAILAND NIDA Center for Research & Development of Disaster Prevention & Management, School of Social and Environmental Development, National Institute of Development Administration (NIDA), 118 Moo 3, Sereethai Road, Klong-Chan, Bangkapi, Bangkok, 10240, THAILAND c

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Faculty of Technology and Environment, Prince of Songkla University Phuket Campus 80 M.1 Kathu, Phuket 83120, THAILAND d

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SKLLQG and Key Lab of Aerosol Chemistry & Physics, Institute of Earth Environment, Chinese Academy of Sciences (IEECAS), Xi’an, 710061, CHINA e

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Department of Meteorology, COMSATS University, Islamabad, PAKISTAN

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*

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Abstract

Corresponding Author: Tele: 00 66 2 727 3113; Fax: 00 66 2 732 0276; Email: [email protected]

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In this study, a combination of the diagnostic binary ratios of PAHs and multivariate

24

descriptive statistics was applied to identify the sources of PAHs in Chilean terrestrial soils.

25

A total of 15 PAHs from the terrestrial soil of 28 locations in three cities of Chile were

26

chemically characterized using gas chromatography mass spectrometry (GC-MS). The total

27

concentrations of twelve likely carcinogenic PAHs were defined as the sum of Phe, An, Fluo,

28

Pyr, B[a]A, Chry, B[b]F, B[k]F, B[a]P, Ind, D[a, h]A and B[g, h, i]P and ranged from 0.0215

29

to 4.37 µg g-1 with an arithmetic mean of 0.618 ± 0.911 µg g-1. The levels of these PAHs

30

were classified as moderate to high compared to World Soils (WS). All sampling stations

31

were dominated by high molecular weight PAHs, four-ring (39.1%) and five-ring (29.6%)

32

PAHs were the most abundant groups in the terrestrial soils of Chile. The PAH diagnostic

1

33

ratios suggested that PAHs are primarily of pyrogenic origin. Further multivariate descriptive

34

statistics (i.e., hierarchical cluster analysis (HCA) and principal components analysis (PCA))

35

identified pyrogenic combustion as the main emission source of PAH contamination in

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Chilean terrestrial soils.

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Keywords: Polycyclic Aromatic Hydrocarbons (PAHs); Terrestrial soils; Chile; diagnostic

38

binary ratio; Hierarchical Cluster Analysis (HCA); Principal Component Analysis (PCA)

39 40

1. INTRODUCTION

41

Over the past few decades, the impact of industrialization on the dispersion of

42

persistent organic pollutants (POPs) has been investigated globally on different continents. In

43

2001, the Stockholm Convention on POPs was officially agreed upon as an international

44

environmental treaty and signed by 128 countries (UNECE, 2019). Polycyclic aromatic

45

hydrocarbons (PAHs) are usually acknowledged as belonging to a group of persistent organic

46

pollutants (POPs) (Pongpiachan et al., 2017a; Cristaldi et al., 2017). These compounds

47

contain two or more benzene rings and PAHs are a class of organic pollutants that includes

48

more than 200 chemicals (Kim et al, 2013; Abdel-Shafy et al., 2016; Zhang et al, 2016;

49

Armstrong et al., 2004). PAHs have been extensively studied given their close connection

50

with adverse health effects and other respiratory diseases (Chalbot et al., 2012; Claxton and

51

Woodall Jr., 2007).

52

PAHs can be generated from both natural and anthropogenic sources. Natural sources

53

include forest fires, volcanoes and crude oil deposits (Abdel-Shafy et al., 2016; Jiao et al.

54

2009). Anthropogenic sources of PAHs in the environment include incomplete combustion

55

from petroleum products, fossil fuels, biofuels or other forms of organic matter (Kim et al.,

56

2008; Zakaria et al., 2002; Davis et al., 2019; Cai et al., 2017). Previous studies reported that

57

PAHs are widely detected in soil (Pongpiachan et al., 2017a, 2018), sediment (Pongpiachan

2

58

et al., 2013b, Tipmanee et al., 2012), atmosphere (Pongpiachan et al., 2013a, 2017b,c),

59

vegetation (Pongpiachan, 2015) and marine organisms (Ke et al., 2017). PAHs are

60

carcinogens, mutagens and teratogens that have toxic effects on organisms through various

61

mechanisms and cause very serious threats to the health and well-being of humans

62

(Pongpiachan et al., 2013a,b; Sette et al., 2013; Yoshimine et al., 2012; Yang et al., 2017;

63

Wang et al., 2015). Therefore, 16 PAHs have been classified as priority pollutants by the US

64

Environmental Protection Agency (US EPA) and are listed in the 1998 Protocols on

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Persistent Organic Pollutants to the Convention on Long Range Transboundary Air Pollution

66

(UNECE, 1998).

67

Soil systems are the most important sinks for PAHs in the environment. High

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concentrations of PAHs have been found in farmland soil throughout the world (Cachada et

69

al., 2012) because PAHs have low water solubility, are readily absorbed by soil particles and

70

tend to accumulate in the soil (Tang et al., 2006; Ping et al., 2007). In addition, numerous

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studies of the correlation of the partition coefficient with soil properties have shown that

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organic carbon content typically yields the most significant correlation (Abdel-Shafy et al.,

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2016; EPRI, 2000); similar results were obtained with the octanol-water partitioning

74

coefficient (Kow), which is related to the solubility of an organic compound in water. Kow is

75

an important parameter in controlling the absorption of dissolved PAHs to terrestrial soils

76

(Schwarzenbach et al., 1993). PAH concentrations are generally greater in urban areas

77

compared with those in rural regions due to comparatively high traffic emissions in cities

78

(Chunhui et al., 2017; Wilcke, 2000; Wang et al., 2007; Wang et al., 2015; Peng et al., 2013).

79

Chile is one of the countries where rapid industrial development is causing

80

environmental problems, especially air pollution. Recently, Chile had set emission standards

81

for power plants (MMA, 2011) along with the use of electricity tariffs as a guide to reduce air

3

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pollution (KAS, 2009; M. Mena-Carrasco, et al., 2012). In addition, other initiatives include

83

establishing standards on passenger and fleet motor vehicle emissions, overhauling the public

84

transportation system and importing cleaner fuels for industrial processes. However, the

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sources of PAHs tend to continuously increase and PAHs are deposited by atmospheric

86

precipitation onto soils (Dong et al., 2012; Abdel-Shafy et al., 2016).

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In this study, terrestrial soil samples were collected in three cities of Chile.

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Qualitative and quantitative analyses of 15 PAHs in the terrestrial soil samples were carefully

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conducted. The main aims of this study were (i) to assess soil concentrations of PAHs in the

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urban and rural areas of Chile and (ii) to chemically characterize the composition of PAHs in

91

the terrestrial soils of Chile using the PAH diagnostic ratios coupled with multivariate

92

descriptive statistical techniques to distinguish the different sources of PAHs.

93

2. MATERIALS AND METHODS

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2.1. Study Areas

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Chile is located in South America between latitude 33°26′S9 and longitude 70°40′W. The

96

western coastline between the Andes Ranges and the Pacific Ocean is 6,435 km long. Chile

97

shares an eastern border with Argentina, a northeastern border with Bolivia and a northern

98

border with Peru. In 2016, the population was estimated to be 18.20 million (CIA, 2019).

99

Most of Chile's industry consists of copper, lithium, minerals, foodstuffs, fish processing,

100

iron and steel, wood products, transport equipment, cement and textiles (CIA, 2019). In this

101

study, terrestrial soil samples (n = 28) were collected in three cities of Chile, namely, i)

102

Valparaíso (33° 3′ 0″ S, 71° 37′ 0″ W) is a major city and is one of the South Pacific’s most

103

important seaports. It has an area of 401.6 km2. For sampling stations of Valparaíso, most

104

located near main roads and community resources as shown in Fig. 1(a). ii) Santiago

105

Metropolitan Region (33°26′16″S, 70°39′01″W) is located in the central valley of the

4

106

country, it covers an area of 15,403.2 km2. Santiago is Chile's smallest division by area, but

107

it is the most densely populated region with a population of over 7 million. Santiago has

108

many vehicles and industries. In 2001, the Journal Science ranked Santiago the second most

109

polluted city after Mexico City because Santiago is located in a watershed land between the

110

coastline and high mountains, which blocks the spread of industrial pollution, pollution from

111

vehicles (Rutllant and Garreaud, 1995; Garcia-Chevesich et al., 2014). Most of the sampling

112

stations in Santiago are located next to the subway, which is the main traffic route in the city

113

as shown in Fig. 1(b). iii) Punta Arenas (53° 10′ 0″ S, 70° 56′ 0″ W) is located in southern

114

Chile on the Brunswick Peninsula, north of the Strait of Magellan. It has a total area of

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17,846.3 km2. Punta Arenas is an important city due to its maritime traffic. It is a portal city

116

supporting shipping trades traveling to the west coasts of South and North America. This

117

study, the sampling stations of Punta Arenas were collected near the beach road, which near

118

the jetty as shown in Fig. 1(c), from 5th January 2016 to 8th February 2016 at the depth of 0 -

119

2 cm. The sampling site coordinates are given in Table 1.

120 121 122 123 124 125 126 127 128 129 130

5

131 132

(a) 133 134 135 136 137 138 139

(b)

141 142 143

Pacific Ocean

140

144 145 146 147

(c)

148 149 150 151 Legend 152 Sampling point 153 154 155

Fig. 1. Sampling site locations in Santiago, Valparaíso and Punta Arenas, Chile.

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156

Table 1. Sampling locations of the terrestrial soil samples collected in Chile (UTM system). Location (UTM) Sample Name

C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28

Date of Collection

5-Jan-2016 5-Jan-2016 5-Jan-2016 5-Jan-2016 5-Jan-2016 5-Jan-2016 5-Jan-2016 7-Feb-2016 7-Feb-2016 7-Feb-2016 7-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016 8-Feb-2016

X

Y

37310 37273 37210 37169 37377 37362 37334 26443 25991 26887 27162 35099 35127 35369 35313 35259 35173 35227 35124 35394 35313 35108 34449 34337 34459 34506 34609 35097

4107781 4107645 4107232 4106235 4108244 4107962 4107843 6343700 6341587 6322723 6322695 6301957 6302331 6280833 6283690 6286886 6289275 6291040 6297432 6298059 6294966 6301189 6297757 6297123 6296557 6297828 6298153 6301156

157 158 159 160

7

Zone

Site Name

19s 19s 19s 19s 19s 19s 19s 19S 19S 19S 19S 19S 19S 19S 19s 19s 19s 19s 19s 19s 19s 19s 19s 19s 19s 19s 19s 19s

Around Punta Arenas Around Punta Arenas Around Punta Arenas Around Punta Arenas Around Punta Arenas Around Punta Arenas Around Punta Arenas Around Valparaiso Around Valparaiso Around Valparaiso Around Valparaiso Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago Around Santiago

161

2.2. Sample Collection and Storage

162

The soil samples were wrapped in pre-cleaned aluminium foil, placed in glass bottles,

163

and kept frozen (−20 °C) until analysis to avoid sample degradation caused by heat, ozone,

164

NO2 and ultraviolet (UV) during transportation (Pongpiachan et al., 2013a, 2017b, c). The

165

samples were then freeze-dried prior to being ground for homogenization. Any wood debris

166

or stones were carefully removed. Then, the soil samples were wrapped in aluminium foil,

167

packed in a plastic bag and then stored at -4 °C until the analysis. Procedural precautions,

168

such as special precautions for trace contaminants of soil sampling, sampling methodology

169

for low concentrations (<200 ng g-1) and quality assurance/quality control (QA/QC),

170

followed the methods of soil sampling outlined in US-EPA Method 5035 (US-EPA, 2002).

171

2.3.PAH Analysis

172

All the organic solvents (i.e., dichloromethane and hexane) were HPLC grade and

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purchased from Fisher Scientific. A standard solution containing a mixture of 15 native

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PAHs [Norwegian Standard (NS 9815: S-4008-100-T): phenanthrene (Phe), anthracene (An),

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fluoranthene

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benzo[b]fluorene

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benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P),

178

benzo[e]pyrene (B[e]P), indeno[1,2,3-cd]pyrene (Ind), dibenz[a,h]anthracene (D[a,h]A) and

179

benzo[g,h,i]perylene (B[g,h,i]P); each 100 µg mL-1 in toluene: unit: 1x1 mL] and a mix of

180

recovery internal standard (IS) PAHs [deuterated-fluorene (d10-Fl) and deuterated-perylene

181

(d12-Per); each 100 µg mL-1 in xylene: unit: 1x1 mL] were purchased from Chiron AS

182

(Stiklestadveine 1, N7041 Trondheim, Norway).

(Fluo),

pyrene

(Pyr),

(11H-B[b]F),

11

H-benzo[a]fluorene

benz[a]anthracene

(B[a]A),

(11H-B[a]F), chrysene

11

H-

(Chry),

183

Approximately 30 g of dried samples were placed in prewashed cellulose (size: 30 × 100

184

mm.). The chemical extraction of the PAHs was performed with a Soxhlet extractor for 8 h

8

185

using DCM as a solvent and internal standards (d10-Fl: Phe, An, Fluo, Pyr, B[a]A, Chry; d12-

186

Per: B[b]F, B[k]F, B[a]P, Ind, D[a,h]A and B[g,h,i]P) were spiked into the sample. In

187

addition, 1 g of activated copper powder was added to remove sulphur. The

188

fractionation/clean-up process was performed strictly in accordance with the method

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described by Gogou et al. (1998). The extractant was passed through a silica gel column,

190

which was activated at 150 °C for 3 h and 5.6:9.4 (v/v) toluene-n-hexanes was used as an

191

eluent. The solution was then purged with nitrogen to almost dryness, then changed into

192

cyclohexane and purged with nitrogen again until the final volume was 100 µl. An aliquot of

193

the solution was further quantified using a gas chromatograph-mass spectrometer (Shimadzu

194

GCMS-QP 2010 Ultra) in the selective ion monitoring mode, which was equipped with a

195

Restek RTX-VRX column (30 m x 0.2 mm i.d. capillary column, 0.5 µm film thickness). All

196

injections (1 µl) were provided through an injector in the splitless mode. The accuracy of the

197

analytical method was evaluated using the standard SRM 1941b, in which the values were

198

under accepted in the certificate of analysis for SRM 1941b as displayed in Table 2. The

199

mean recovery based on the extraction of matrix-matched certified reference materials was in

200

the range of 77–119%. The precision of the procedure, which was calculated as the relative

201

standard deviation of the duplicate samples, was less than 15%. All sample concentrations

202

were calculated using standardized relative response factors that were run with each batch

203

(Pongpiachan et al., 2009a,b; Tipmanee et al., 2012).

204

2.4 Statistical analysis

205

This study, statistical analysis was using Microsoft Excel (Microsoft Inc., USA) and

206

Statistics Package for the Social Sciences (SPSS) software version 22 (SPSS Inc., USA)

207

including Principal Component Analysis (PCA) and Hierarchical cluster analysis (HCA).

208

PCA as a multivariate analytical tool was used to find new components (Principal

209

Components) as a linear combination of the original variables and to separate a little number

9

210

of the prominent principal components to explain the relationship between observed variables

211

(Larsen and Baker, 2003). For this study, to obtain clearer features, the Varimax rotation

212

method was used with Kaiser standard adjustment. While the purpose of using HCA was to

213

group stations based on the relative contents of PAH compounds in the terrestrial soil

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samples. The squared Euclidean distance was used to measure the distance of similarities

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between objects (Fang et al., 2007). The amalgamation of clusters follows the method of

216

Ward’s method (Savinov et al., 2000).

217

Table 2. Accuracy test with SRM 1941b.

n

Reference Valve ng g−1 dry weight

Measuring Valve ng g−1 dry weight

% Accuracy

Phe

8

406±44.0

464±16.0

86.0±4.00

Fluoranthene

Fluo

8

651±50.0

721±45.0

88.0±7.00

Pyrene

Pyr

8

581±39.0

538±34.0

106±7.00

Benzo[a]anthracene

B[a]A

8

335±25.0

289±26.0

114±8.00

Chrysene

Chry

8

291±31.0

336±25.0

83.0±8.00

Benzo[b]fluoranthene

B[b]F

8

453±21.0

480±25.0

94.0±6.00

Benzo[k]fluoranthene

B[k]F

8

225±18.0

229±19.0

100±8.00

Benzo[e]pyrene

B[e]P

8

325±25.0

321±10.0

101±3.00

Ind

8

341±57.0

291±14.0

115±4.00

B[g,h,i]P

8

307±45.0

267±17.0

113±6.00

Abbreviati on

Phenanthrene

SRM

Indeno[1,2,3-cd]pyrene Benzo[g,h,i]perylene 218 219

220

221

222

10

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3. RESULTS AND DISCUSSION

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3.1 PAH contamination in Chilean terrestrial soils

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The total concentrations of 15 PAHs (∑15PAHs: a sum of Phe, An, Fluo, Pyr, 11H-

226

B[a]F, 11H-B[b]F, B[a]A, Chry, B[b]F, B[k]F, B[e]P, B[a]P, Ind, D[a,h]A and B[g,h,i]P) in

227

the Chilean terrestrial soil samples were qualitatively and quantitatively assessed. The total

228

concentration of the PAHs (∑15PAHs) ranged from 0.0234 to 4.82 µg g-1 in dry weight with

229

an average of 0.681 ± 1.00 µg g-1. In this study, the total concentration of twelve probable

230

carcinogenic PAHs was defined as the sum of Phe, An, Fluo, Pyr, B[a]A, Chry, B[b]F,

231

B[k]F, B[a]P, Ind, D[a, h]A and B[g, h, i]P (i.e., ∑12PAHs). Here, the ∑12PAH vales in the

232

Chilean terrestrial soils ranged from 0.0215 to 4.37 µg g-1 dry weight with an arithmetic

233

mean of 0.618 ± 0.911 µg g-1 dry weight. For the total concentration (∑12PAHs) of Punta

234

Arenas were range 0.0215 to 0.107 µg g-1 dry weight with an average of 0.0591± 0.0356 µg

235

g-1, The Valparaiso ranged from 0.0774 to 0.738 µg g-1 dry weight with an average of 0.397 ±

236

0.290 µg g-1 dry weight and Santiago was in the range 0.0663 to 4.37 µg g-1 dry weight with

237

an arithmetic mean of 0.901± 1.07 µg g-1 dry weight. Overall, the results have shown that the

238

trend of PAH concentrations was high at the storage stations of Santiago, followed by

239

Valparaiso and Punta Arenas, respectively (see Fig. 2a).

240

Table 3 shows the arithmetic mean and standard deviation of the PAHs in Chilean

241

terrestrial soils and World Soils (WS). As displayed in Table 3, the total concentrations of the

242

∑12PAHs in Chile were lower than those in the soils collected in the Northern Region of

243

France (1853 µg g-1), Dilovasi, Turkey (0.949 µg g-1 ) and Northern Part of Poland (1.49 µg

244

g-1) (Lors et al., 2012; Cetin, 2016; Melnyk et al., 2015). However, the concentration of the

245

PAHs in Chile was 190- and 19-fold higher than those of King George Island, Antarctica

246

(0.00325 µg g-1) and Koh Samed Island, Thailand (0.0327 µg g-1), respectively (Pongpiachan

11

247

et al., 2017, 2018). Here, the ∑12PAHs showed the highest value at C27 followed by C28,

248

C13, C26 and C24, with values of 4.37 µg g-1, 2.29 µg g-1, 1.61 µg g-1, 1.20 µg g-1 and 1.05

249

µg g-1, respectively. Table 3. Statistical descriptions of the PAH concentrations (µg g-1 dry weight) collected in Chile compared with WS.

250 251

Compound Phe An Fluo Pyr B[a]A Chry B[b]F B[k]F B[a]P Ind D[a,h]A B[g,h,i]P Σ12PAHs 3-Ring PAHs 4-Ring PAHs 5-Ring PAHs 6-Ring PAHs 3-Ring PAHs/4-Ring 4-Ring PAHs/5-Ring 5-Ring PAHs/6-Ring 252 253 254 255 256 257 258 259

Chilea Aver 0.0758 0.00943 0.0846 0.0892 0.0383 0.0709 0.0792 0.0293 0.0545 0.0360 0.00883 0.0424 0.618 0.0852 0.283 0.171 0.0785 0.000300 0.00165 0.00219

Stdev 0.0937 0.0127 0.143 0.154 0.0689 0.100 0.125 0.0424 0.0982 0.0531 0.0120 0.0560 0.911 0.106 0.454 0.271 0.108

a

WSb Aver 52.6 17.0 37.1 22.7 9.37 8.57 7.49 3.00 6.95 0.990 4.01 3.69 173 69.6 77.8 18.4 7.70 0.000890 0.00422 0.00239

Stdev 173 55.8 114 64.7 24.3 21.7 17.9 7.50 17.5 2.27 9.40 8.97 511 229 223 45.0 18.3

The average concentrations of PAHs in Chile from this study. WS is the average of PAHs in soils collected in King George Island, Antarctica (Pongpiachan et al., 2017), Koh Samed Island, Thailand (Pongpiachan et al., 2018), Dilovasi, Turkey (Yurdakul el al., 2019), Dalian, China (Wang et al., 2009), Northern Guangdong Province of China (Wang et al., 2012), Beijing, China (Sun et al., 2012), Dhanbad District of Jharkhand, India (Suman et al., 2016), Northern Part of Poland (Melnyk et al., 2015), Northern Region of France (Lors et al., 2012), Ulsan, Korea (Kwon and Choi, 2014), Neuves-Maisons, France (Laurent et al., 2012), Australia (Juhasz et al., 2016) and Dilovasi, Turkey (Cetin, 2016). b

260

12

261

PAHs can be classified according to their molecular structure by categorization into

262

four groups: three-ring PAHs (i.e., Phe and An), four-ring PAHs (i.e., Fluo, Pyr, B[a]A and

263

Chry), five-ring PAHs (i.e., B[b]F, B[k]F, B[a]P and D[a,h]A) and six-ring PAHs (i.e., Ind

264

and B[g,h,i]P). The results showed that the four-ring PAHs (39.1%) and five-ring PAHs

265

(29.6%) are the most abundant groups in the terrestrial soils of Chile and the abundance

266

ranged from 14% to 60% and 12% to 73%, respectively (see Fig. 2b). Sampling points C26

267

and C20 exhibited the highest levels of four ring compounds, corresponding to 60% and 59%

268

of the total PAHs, respectively. The highest concentrations of the total PAHs were observed

269

at stations C26 and C20 because the sampling points are located near the centre of Santiago,

270

which is close to a university, school, amusement park and racing tracks. This finding is

271

consistent with the research of Wang et al. (2007) that reported that the highest concentration

272

of PAHs was found in areas with high traffic followed by park/residential sites and suburban

273

sites. In contrast, rural sites contain the lowest levels of PAHs.

274

Among the low and middle molecular weight PAHs (LMW PAHs: 3 aromatic ring

275

PAHs; MMW PAHs: 4 aromatic ring PAHs), the highest concentrations of PAHs were

276

observed for Pyr, Fluo, Chry and Phe with average concentrations of 0.753 µg g-1, 0.676 µg

277

g-1, 0.396 µg g-1 and 0.336 µg g-1, respectively. These LMW PAHs are derived from the

278

burning of coal (Ravindra et al., 2007) and other biomass at low temperatures. In contrast,

279

Miguel et al. (1998) found that diesel trucks were the primary source of LMW PAHs. In

280

contrast, materials, such as oil or gasoline, that burn at high temperatures and light-duty

281

gasoline vehicles produce middle and high molecular weight PAHs (HMW PAHs: 5-6

282

aromatic ring PAHs) (Miguel et al., 1998). In this study, HMW PAHs had average

283

percentage contributions of 30.0% and 14.0% for the five and six aromatic ring PAHs,

284

respectively. However, the diagnostic binary ratios of the 3-Ring PAHs/4-Ring PAHs (i.e.,

13

285

0.30), 4-Ring PAHs/5-Ring PAHs (i.e., 1.65) and 5-Ring PAHs/6-Ring PAHs (i.e., 2.19)

286

obtained from this study are relatively low compared with those of WS (see Table 3). We

287

observed that the LMW PAHs and MMW PAHs are the most abundant PAHs in Chilean

288

soils. Several previous studies have suggested that Pyr, Phe and Fluo accounted for

289

approximately 75.0% of the total PAHs emissions from heavy-duty diesel vehicles (HDDVs),

290

including diesel trucks, ships and trains (Zheng et al.,2017). In addition, HDDVs were the

291

major source of 3-benzene ring PAHs, such as Fluo and Pyr (Miguel et al., 1998; Marr et al.,

292

1999, 2006; Ravindra et al., 2007, 2008). In contrast, light-duty vehicles (LDPVs), including

293

passenger cars, sport utility vehicles and minivans, were the major source of 4- to 5-benzene

294

ring PAHs (Marr et al., 2006; Ravindra et al., 2007, 2008). Zheng et al. (2017) found that

295

LDPVs are the major contributors of HMW PAHs.

296

297

298

299

300

301

302

303

304

305

14

306

a 307

308

309

310

311

312

313

314 Punta Arenas

Valparaiso

Santiago

Valparaiso

Santiago

315

b 316

317

318

319

320

321 322 323 324

Punta Arenas

Fig. 2. (a) Concentration of the PAHs and (b) Distribution patterns of percentage contribution (%) and classification of 3-6 ring PAHs in terrestrial soil of Chile.

325

15

326

3.2 Source identification based on diagnostic PAH isomer ratios

327

PAHs are the most stable form of hydrocarbons; the characteristics of PAH patterns

328

in the soils are subject to different emission sources. The diagnostic binary ratios of PAHs

329

can be used to identify potential emission sources that can be further categorized into

330

pyrolytic or petrogenic processes (Yunker et al., 2002; Davis et al., 2019; Chen et al., 2012;

331

Wang et al., 2018; Duodu et al., 2017; Tobiszewski et al., 2012). A pyrolytic process

332

involves the incomplete combustion of biomass or fossil fuels (Manahan, 1994), while a

333

petrogenic process involves the slow maturation of organic substances as a result of a leakage

334

of crude oil (Abdel-Shafy et al., 2016).

335

Five diagnostic binary ratios of PAH isomer pairs, namely, An/(An+Phe),

336

Fluo/(Fluo+Pyr), B[a]A/(B[a]A+Chry), Ind/(Ind+B[g,h,i]P) and B[a]P/B[g,h,i]P, were used

337

to determine the source of PAHs in Chilean terrestrial soils. The five diagnostic PAH ratios

338

used in previous studies to classify the pyrogenic and petrogenic sources. A An/(An+Phe)

339

ratio < 0.10 indicates pyrogenic sources, such as incomplete combustion, whereas a ratio >

340

0.10 indicates petrogenic sources, such as petroleum products and oil spill (Budzinski et al.,

341

1997; Tobiszewski et al., 2012). A Fluo/(Fluo+Pyr) ratio of less than 0.40 indicates unburned

342

petroleum. In contrast, values ranging from 0.40 to 0.50 indicate mixed sources of various

343

types of liquid fossil fuel combustion (e.g., gasoline and crude oil) (Torre-Roche et al., 2009).

344

A Fluo/(Fluo+Pyr) ratio greater than 0.50 indicates pyrogenic sources of grass, wood, or coal

345

combustion (Yunker et al., 2002). In addition, Ravindra et al. (2008) also reported that a

346

Fluo/(Fluo+Pyr) ratio < 0.50 indicates gasoline emissions, whereas a ratio > 0.50 indicates

347

diesel emissions. A B[a]A/(B[a]A Chry) ratio < 0.20 indicates petrogenic sources, a ratio

348

from 0.20 to 0.35 indicates either petroleum or combustion (mixed sources) and a ratio > 0.35

349

indicates combustion. A Ind/(Ind B[g,h,i]P) ratio < 0.20 likely indicates petroleum, a ratio

16

350

from 0.20 to 0.50 indicates petroleum combustion and a ratio > 0.50 indicates grass, wood

351

and coal combustion (Yunker et al., 2002). A B[a]P/B[g,h,i]P ratio < 0.60 indicates non-

352

traffic emissions and a ratio > 0.60 signals traffic emissions.

353

Cross-plots of the ratio between Fluo/(Fluo+Pyr) versus B [a] A/(B [a] A+Chry) (see

354

Fig. 3) are useful to identify the potential sources of the PAHs in soil from Chile. The results

355

showed that most of the PAHs in the terrestrial soil samples originated from the mixed

356

sources from the incomplete combustion of coal, fuel and oil burning. In 2016, the primary

357

energy consumption for Chile was 1.52 quadrillion btu, which increased from 0.93

358

quadrillion btu in 1997 to 1.52 quadrillion btu in 2016, growing at an average annual rate of

359

2.63%. The energy consumption in Chile is dominated by fossil fuels with coal, oil and gas

360

accounting for the majority of total primary energy (73.4%) and biofuels accounting for

361

20.5% (IEA, 2017)

362

The cross plots of Fluo/(Fluo+Pyr) versus An/(An+Phe) divided the samples into two

363

major contributors, namely, mixed and combustion sources, as shown in Fig. 3. The

364

horizontal axis with a Fluo/(Fluo+Pyr) ratio of 0.40 indicates mixed pyrogenic sources and

365

fossil fuel combustion as plausible emission sources. However, the B[a]A/(B[a]A+Chry) and

366

Ind/(Ind+ B[g,h,i]P) ratio indicated that most of the PAHs in the soil samples originated from

367

pyrogenic sources of burning petroleum. In addition, the B[a]P/B[g,h,i]P ratio indicates that

368

vehicle exhaust is the main contributor of PAHs, in which the highest value is noted in S26.

369

The results of the use of the isomer show that most PAHs in the Chilean terrestrial

370

soil samples are primarily derived from combustion, which is consistent with the increasing

371

trend of vehicles and industrial plants in Chile (Economic Diplomacy Division, 2018). It is

372

also crucial to emphasize that some heavy smog clouded the capital of Chile in 1996. The

373

main contributors to the accumulation of air pollution are automobiles, buses, trucks, power

17

374

plants, boilers, industrial processes, foundries, metal processes, biomass burning and

375

combustion of agricultural fires (Garcia-Chevesich et al., 2014).

376

377

378

379

a

b

c

d

380

381

382

383

384

385 386 387 388

Fig. 3. Cross-plot for the ratios: (a) B[a]A/(B[a]A+ Chry) versus Fluo/(Fluo+Pyr), (b) An/(An+Phe) versus Fluo/(Fluo+Pyr), (c) B[a]P/B[g,h,i]P versus Ind/(Ind+ B[g,h,i]P) and (d) Ind/(Ind+ B[g,h,i]P) versus B[a]A/(B[a]A+ Chry) in the soils from Chile

389

The growing industry in the main cities of Chile, especially Santiago, is also the main

390

cause of the release of PAHs into the environment and their accumulation in terrestrial soil

391

samples. Nevertheless, it is important to note that the isomer pair ratios of the entire dataset

392

may unintentionally allow for misinterpretation when the mixtures of PAH sources are

393

analysed. To avoid any systematic errors that could occur from applying the unstable isomer

394

pair ratios (i.e., 3-4 ring PAHs), one should employ the HMW PAHs ratios for analysis

18

395

because they are much more stable in solving the ratio change problems (Chunhui et al.,

396

2017).

397

3.3 Source estimation from hierarchical cluster analysis (HCA)

398

399

400

401

402

403 404 405 406

Fig. 4. Hierarchical cluster analysis (HCA) of 13 individual PAHs in Chilean soil.

407

HCA is an algorithm that can be used to categorize comparatively smaller sample

408

numbers (i.e., n=200) without pre-preparing the data (Dachs et al., 1999). In this study, HCA

409

was performed to identify the homogeneous groups of individual PAHs in the Chilean soil

410

samples. The results show that the major PAHs are divided into three groups of 13

411

individuals. The first group contains An, D[a,h]A, B[a]A and B[k]F, which originated from

412

pyrogenic sources (Ravindra et al., 2008; Khalili et al., 1995). This finding is consistent with

413

research from Khalili et al. (1995) that demonstrated that 2- to 5-ring PAHs are released from

414

motor vehicle, diesel engine, petrol engine, coke oven and wood combustion. The second

415

group consists of Fluo, Pyr, Chry and Phe, which are 4-ring PAHs. This group typically

416

originated from the use of petroleum products, heavy-duty diesel vehicles and industrial

19

417

factories using fossil fuels (Yang et al., 1998; Maliszewska-Kordybach, 1999; Wild et al.,

418

1995; Tipmanee et al., 2012). The last group consists of B[e]P, Ind, B[g,h,i]P, B[a]P and

419

B[b]F, which typically originated from motor vehicles (Smith and Harrison, 1998; Ravindra

420

et al., 2007), asphalt (Ravindra et al., 2007, 2008), rubber tire abrasion (Marchesani et al.,

421

1970; Rogge et al., 1993), brake linings and road dust ( Ahrens and Depree, 2010;

422

Marchesani et al., 1970; Rogge et al., 1993; Boulter, 2005) as shown in Fig. 4.

423

As expected, the HCA results suggest a pyrogenic source of PAHs. Although most

424

PAHs are emitted into the atmosphere, PAHs can be absorbed by particles that settle by wet

425

and dry deposition into terrestrial soils. In addition, cluster analysis results of the 28

426

individual sampling stations revealed two major groups as shown in Fig. 5.

427

The first group contains stations C1, C2, C3, C4, C5, C6, C7, C10 and C12. All of

428

these stations have distinct characteristics compared with other areas and almost all of the

429

stations in this group are located in Punta Arenas, Chile. The sampling station is located near

430

the Punta Arenas harbour, where it is used as a coaling station between the Atlantic and

431

Pacific oceans. However, currently, the Punta Arenas harbour is mostly used by scientific

432

expeditions and tourism cruises to Antarctica. The harbour also serves as a hub to other

433

regions for many cruise lines. Therefore, this group represents PAHs arising from diesel fuel

434

of large engines of larger ships and ferries. The HCA results are consistent with previous

435

reports on PAH contamination that found that LMW PAHs are high in this area. The nearby

436

group has similar characteristics and consists of C11, C14, C16, C17, C18, C21, C23 and

437

C25. This finding also indicates the influence of incomplete combustion of fuels from light-

438

duty gasoline vehicles and petrol engine. This group is as expected because it is located in the

439

centre of Santiago, which has numerous traffic problems. These areas were carefully chosen

20

440

as study sites because they are differently affected by sources of PAHs. Santiago is Chile's

441

smallest city by area, but it is the most densely populated regions and has traffic problems.

442 443 444 445

Fig. 5. Hierarchical cluster analysis (HCA) of 28 individual sampling stations in Chilean terrestrial soils.

446

3.4 Source identification by principal components analysis (PCA)

447

In this study, PCA was used to identify the source of 13 PAHs in 28 terrestrial soil

448

samples. As shown in Table 4, scalable data were explained by two eigenvectors–principal

449

components that control 94.5% of the variability of the data. The first principal component

450

(PC1) explained 53.1% of the total variance. This factor is contributed by the strong loading

451

of B[a]A, B[b]F, B[e]P, B[a]P, Ind, D[a,h]A and B[g,h,i]P, which, except for B[a]A, are 5-

452

to 6-ring PAHs. Numerous sources, such as the incomplete combustion and pyrolysis of fuel,

453

road paving asphalt, gasoline, and road dust, have been widely considered to be the main

454

sources of 5- to 6-ring PAHs (Miguel et al., 1998). The PCA results are related to the HCA

455

results of the 28 sampling stations, indicating that PC1 is the entire sampling station located

456

in Santiago and some stations of Valparaiso (see Fig. 6). Thus, it seems reasonable to

21

457

interpret PC1 might be representative of the incomplete combustion of fuels from light-duty

458

gasoline vehicles, road paving asphalt, gasoline or road dust (Smith and Harrison, 1998;

459

Ravindra et al., 2007) caused by traffic congestion in the area.

460

Table 4. Rotated component matrix of 13 PAHs from the Chilean terrestrial soilsa. Principle component (PC) PC1 PC2 Phe 0.34 0.92 An 0.52 0.81 Fluo 0.68 0.72 Pyr 0.66 0.72b B[a]A 0.64 0.73 Chry 0.56 0.67 B[b]F 0.42 0.90 B[k]F 0.64 0.72 B[e]P 0.51 0.85 B[a]P 0.53 0.80 Ind 0.46 0.87 D[a,h]A 0.50 0.84 B[g,h,i]P 0.45 0.83 Total variance (%) 53.1 41.3 Cumulative (%) 53.1 94.5 a Rotation method: Varimax with Kaiser normalization. b Bold number: Loading value greater than 0.7 (heavy loading) PAH composition

461 462 463 464

.In contrast, the second factor (PC2) explained 41.3% of the total variance. The

465

weight of the factor was predominately composed of Phe, An, Fluo, Pyr and B[k]F. The low

466

molecular weight PAHs with 3-4 rings (i.e., Phe, An, Fluo and Pyr) were predominant in PC2

467

and these PAHs typically originate from diesel emissions from transportation vehicles, such

468

as ships, truck and trains (Ravindra et al., 2007; Kulkarni and Venkataraman, 2000; Ho et al.,

469

2002). Furthermore, the PC2 results are related to the HCA results of the 28 sampling

470

stations, indicating that PC2 is the entire sample station in Punta Arenas (see Fig. 6), which is

471

near the port . In addition, Fang et al. (2006) showed that the high loadings of Fluo and Pyr

472

demonstrated the incomplete combustion of fuel oil. As a consequence, it seems rational to

22

473

conclude that PC2 is a representative of the incomplete combustion of oil fuel from land

474

vehicles and water vehicles.

475 476 477

Fig. 6. Score plot of variables component 1 versus component 2 for the terrestrial soils samples of Chile by PCA.

478 479

The results of the identification of the sources of polycyclic aromatic hydrocarbons in

480

terrestrial soils showed that incomplete combustion from the use of fuel is a key source of

481

polycyclic aromatic hydrocarbons in Chile. We expect that this study will benefit the

482

environmental planning management of Chile, as well as that of many other countries, in the

483

future. The sample study of Keyte et al. (2016) reports a large (~85%) decline in PAH

484

concentrations between the 1992 and 2012 measurements in the Queensway Road Tunnel

485

(QT) in Birmingham, U.K., due to increasingly stringent EU vehicle emissions legislation

486

and the introduction of catalytic converters in the U.K.

23

487

4. Conclusions

488

PAH components in soil were analysed via multivariate descriptive statistical

489

techniques. The levels of PAH pollution (∑15PAHs) in Chile range from 0.0234 to 4.82µg g-1

490

dry weight, which is a low to moderate concentration compared to results from previous

491

studies. The determination of the source of PAHs in Chilean soils depends on the distribution

492

of individual compounds in combination with the application of diagnostic ratios, HCA and

493

PCA. The PAH individual distribution results indicate that pyrogenic sources are the main

494

source of PAH in Chilean soils because HMW PAHs with 4 rings (39.1%) and 5 rings

495

(29.6%) are the most abundant. The results from the diagnostic PAH isomer ratios suggested

496

that the PAHs in most soils in all the sampling stations originated from the incomplete

497

combustion of petroleum products and the B[a]P/B [ghi] ratio showed that most of the PAHs

498

in the soil samples originated from traffic emissions. The results of soil PAH composition

499

analysis in combination with the use of the multivariate descriptive statistical techniques

500

clearly support our hypothesis that PAHs were transferred from a potential source in the air

501

and eventually deposited onto the surface soil.

502

Acknowledgements

503

The authors acknowledge the Information Technology Foundation under the Initiative of Her

504

Royal Highness Princess Maha Chakri Sirindhorn, Polar Research Project under the

505

Initiatives of Her Royal Highness Princess Maha Chakri Sirindhorn, National Science and

506

Technology Development Agency (NSTDA), Chinese Arctic and Antarctic Administration

507

and T. C. Pharmaceutical Industries Co., Ltd. for supporting this study. The authors would

508

like to thank the Faculty of Environmental Management, Prince of Songkla University and

509

the Division of Environmental Science and Technology, Faculty of Science and Technology,

24

510

Rajamangala University of Technology Phra Nakhon for providing the facility to conduct this

511

study.

512

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•

Pyrogenic sources are the main source of PAH in Chilean soils.

•

∑15PAHs in Chile range from 0.0234 to 4.82 µg g-1 in dry weight.

•

Traffic emissions play an important role in governing PAH contents.

1 Author declaration [Instructions: Please check all applicable boxes and provide additional information as requested.] 1. Conflict of Interest Potential conflict of interest exists: We wish to draw the attention of the Editor to the following facts, which may be considered as potential conflicts of interest, and to significant financial contributions to this work: The nature of potential conflict of interest is described below:

No conflict of interest exists. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

2. Funding Funding was received for this work. All of the sources of funding for the work described in this publication are acknowledged below: Her Royal Highness Princess Maha Chakri Sirindhorn, Polar Research Project under the Initiatives of Her Royal Highness Princess Maha Chakri Sirindhorn, National Science and Technology Development Agency (NSTDA), Chinese Arctic and Antarctic Administration, and T. C. Pharmaceutical Industries Co., Ltd.

No funding was received for this work.

3. Intellectual Property

2 We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.

4. Research Ethics We further confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. IRB approval was obtained (required for studies and series of 3 or more cases) Written consent to publish potentially identifying information, such as details or the case and photographs, was obtained from the patient(s) or their legal guardian(s).

5. Authorship The International Committee of Medical Journal Editors (ICMJE) recommends that authorship be based on the following four criteria: 1. Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; AND 2. Drafting the work or revising it critically for important intellectual content; AND 3. Final approval of the version to be published; AND 4. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All those designated as authors should meet all four criteria for authorship, and all who meet the four criteria should be identified as authors. For more information on authorship, please see http://www.icmje.org/recommendations/browse/roles-andresponsibilities/defining-the-role-of-authors-and-contributors.html#two. All listed authors meet the ICMJE criteria. We attest that all authors contributed significantly to the creation of this manuscript, each having fulfilled criteria as established by the ICMJE. One or more listed authors do(es) not meet the ICMJE criteria. We believe these individuals should be listed as authors because:

3 [Please elaborate below]

We confirm that the manuscript has been read and approved by all named authors. We confirm that the order of authors listed in the manuscript has been approved by all named authors.

6. Contact with the Editorial Office The Corresponding Author declared on the title page of the manuscript is: Prof. Dr. Siwatt Pongpiachan Director of NIDA Center for Research & Developmentof Disaster Prevention & ManagementSchool of Social and Environmental Development, National Institute of Development Administration(NIDA), 118 Moo3, Sereethai Road, KlongChan, Bangkapi,Bangkok 10240 THAILAND This author submitted this manuscript using his/her account in EVISE. We understand that this Corresponding Author is the sole contact for the Editorial process (including EVISE and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that the email address shown below is accessible by the Corresponding Author, is the address to which Corresponding Author’s EVISE account is linked, and has been configured to accept email from the editorial office of American Journal of Ophthalmology Case Reports: Email: [email protected] Someone other than the Corresponding Author declared above submitted this manuscript from his/her account in EVISE: [Insert name below]

We understand that this author is the sole contact for the Editorial process (including EVISE and direct communications with the office). He/she is responsible for communicating with the other authors, including the Corresponding Author, about progress, submissions of revisions and final approval of proofs.

1 Author declaration [Instructions: Please check all applicable boxes and provide additional information as requested.] 1. Conflict of Interest Potential conflict of interest exists: We wish to draw the attention of the Editor to the following facts, which may be considered as potential conflicts of interest, and to significant financial contributions to this work: The nature of potential conflict of interest is described below: No conflict of interest exists. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

2. Funding Funding was received for this work. All of the sources of funding for the work described in this publication are acknowledged below: The authors acknowledge the Information Technology Foundation under the Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn, Polar Research Project under the Initiatives of Her Royal Highness Princess Maha Chakri Sirindhorn, National Science and Technology Development Agency (NSTDA), Chinese Arctic and Antarctic Administration and T. C. Pharmaceutical Industries Co., Ltd. for supporting this study. No funding was received for this work.

3. Intellectual Property We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.

2 4. Research Ethics We further confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. IRB approval was obtained (required for studies and series of 3 or more cases) Written consent to publish potentially identifying information, such as details or the case and photographs, was obtained from the patient(s) or their legal guardian(s).

5. Authorship The International Committee of Medical Journal Editors (ICMJE) recommends that authorship be based on the following four criteria: 1. Substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; AND 2. Drafting the work or revising it critically for important intellectual content; AND 3. Final approval of the version to be published; AND 4. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All those designated as authors should meet all four criteria for authorship, and all who meet the four criteria should be identified as authors. For more information on authorship, please see http://www.icmje.org/recommendations/browse/roles-andresponsibilities/defining-the-role-of-authors-and-contributors.html#two. All listed authors meet the ICMJE criteria. We attest that all authors contributed significantly to the creation of this manuscript, each having fulfilled criteria as established by the ICMJE. 6. Contact with the Editorial Office The Corresponding Author declared on the title page of the manuscript is: Siwatt Pongpiachan Email: [email protected] This author submitted this manuscript using his/her account in EVISE. We understand that this Corresponding Author is the sole contact for the

3 Editorial process (including EVISE and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that the email address shown below is accessible by the Corresponding Author, is the address to which Corresponding Author’s EVISE account is linked, and has been configured to accept email from the editorial office of American Journal of Ophthalmology Case Reports: [email protected]

We understand that this author is the sole contact for the Editorial process (including EVISE and direct communications with the office). He/she is responsible for communicating with the other authors, including the Corresponding Author, about progress, submissions of revisions and final approval of proofs.

We the undersigned agree with all of the above. Woranuch Deelaman, Siwatt Pongpiachan, Danai Tipmanee, Chomsri Choochuay, Natthapong Iadtem, Oramas Suttinun, Qiyuan Wang, Li Xing, Guohui Li, Yongming Han, Muhammad Zaffar Hashmi, Junji Cao