Analysis of urinary metabolites of polycyclic aromatic hydrocarbons and cotinine in pooled urine samples to determine the exposure to PAHs in an Australian population.

Journal Pre-proof Analysis of urinary metabolites of polycyclic aromatic hydrocarbons and cotinine in pooled urine samples to determine the exposure to PAHs in an Australian population. Phong K. Thai, Andrew P.W. Banks, Leisa-Maree L. Toms, Phil M. Choi, Xianyu Wang, Peter Hobson, Jochen F. Mueller PII:

S0013-9351(19)30845-X

DOI:

https://doi.org/10.1016/j.envres.2019.109048

Reference:

YENRS 109048

To appear in:

Environmental Research

Received Date: 2 August 2019 Revised Date:

13 December 2019

Accepted Date: 13 December 2019

Please cite this article as: Thai, P.K., Banks, A.P.W., Toms, L.-M.L., Choi, P.M., Wang, X., Hobson, P., Mueller, J.F., Analysis of urinary metabolites of polycyclic aromatic hydrocarbons and cotinine in pooled urine samples to determine the exposure to PAHs in an Australian population., Environmental Research (2020), doi: https://doi.org/10.1016/j.envres.2019.109048. 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. © 2019 Published by Elsevier Inc.

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Analysis of urinary metabolites of polycyclic aromatic hydrocarbons and

3

cotinine in pooled urine samples to determine the exposure to PAHs in an

4

Australian population.

5

Phong K Thaia,1*, Andrew P W Banks a,1, Leisa-Maree L Tomsb, Phil M Choia, Xianyu Wanga,

6

Peter Hobsonc, Jochen F Mueller a

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a

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(QAEHS), 20 Cornwall Street, Woolloongabba, QLD 4102, Australia;

The University of Queensland, Queensland Alliance for Environmental Health Sciences

10

b

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Queensland University of Technology, Brisbane, QLD, Australia

12

c

Sullivan Nicolaides Pathology, Taringa, QLD, Australia;

1

These authors contributed equally to this paper

School of Public Health and Social Work and Institute of Health and Biomedical Innovation,

13 14 15 16 17

*Corresponding author:

18

Phong Thai, [email protected];

19 20 21 22

1

23 24

Abstract

25

Our previous biomonitoring study of hydroxylated polycyclic aromatic hydrocarbons (OH-

26

PAHs) in a population in Australia found high levels of 1-naphthol, a metabolite of both

27

naphthalene and carbaryl, in some adult samples. Here, we conducted a follow-up study to

28

collect and analyse pooled urine samples, stratified by age and sex, from 2014 to 2017 using a

29

GC-MS method. Geometric mean concentrations of 1-hydroxypyrene, the most common

30

biomarker of PAH exposure, were 100 and 120 ng/L urine in 2014-2015 and 2016-2017,

31

respectively. The concentrations of most OH-PAHs in this study except 1-naphthol are in line

32

with those reported by biomonitoring programs in the US and Canada. In general, concentrations

33

of OH-PAHs are lower in samples from small children (0-4 years) and school-aged children (5-

34

14 years) compared with samples from the older age groups, except for some cases in the recent

35

monitoring period. The concentrations of 1-naphthol in some adult samples of both sexes are

36

very high, which is consistent with our previous findings. Such high concentrations of 1-

37

naphthol together with the high 1-naphthol/2-naphthol ratio suggest potential exposure to the

38

insecticide carbaryl in this population but other exposure sources and different rates of

39

naphthalene metabolism should also be investigated.

40 41

Keywords:

OH-PAHs;

42

biomonitoring;

urinary

metabolites;

1-NAP/2-NAP

ratio;

children;

adults;

43

2

44

1. Introduction

45

Polycyclic aromatic hydrocarbons (PAHs) are a class of hazardous pollutants produced during

46

the incomplete combustion of organic materials either naturally (e.g. bush fires) or

47

anthropogenically (e.g., vehicular emissions, power plants, wood smoke), with several PAHs

48

classified as probable human carcinogens (IARC, 2010; Kim et al., 2013). Epidemiological

49

studies have linked exposure to PAHs with childhood obesity and behavioural changes (Perera et

50

al., 2014, Rundle et al., 2012), and urinary metabolites of PAHs have been associated with

51

childhood obesity (Scinicariello and Buser, 2012). There is also a potential relationship between

52

exposure to PAHs and the risk of alteration of the immune system (Walker et al., 2013). Due to

53

the adverse health effects of PAH exposure, regular monitoring of PAHs in the population will

54

help to undestand the risk from PAHs in the general population.

55

Previously, we have presented the first assessment of exposure to PAHs in an Australian

56

population including small children (0-4 years) through monitoring of urinary mono-

57

hydroxylated PAHs (OH-PAHs) in pooled urine samples (Thai et al., 2016). Biomonitoring

58

provides an aggregate estimate of exposure to low molecular weight PAHs (up to 4 rings),

59

integrating exposures from all sources and pathways, i.e. inhalation, ingestion, and dermal

60

absorption (Sexton et al. 2004). We found that the concentrations of most urinary OH-PAHs

61

measured in samples collected in Australia are similar to those reported for developed countries

62

(e.g. the US, Germany, Canada) and lower than those reported for some developing countries

63

(e.g. China, Vietnam) except for 1-naphthol (1-NAP), which has, to our knowledge, the highest

64

geometric mean value among all population studies reported in the literature (Thai et al., 2016).

65

Such high levels of 1-NAP could indicate exposure to the insecticide carbaryl, which is

66

metabolized to 1-NAP in the human body (Meeker et al., 2007).

67

The findings above provided a snapshot understanding of the population exposure to PAHs and

68

potentially carbaryl but follow-up monitoring is needed to assess whether the high level of 1-

69

NAP is consistent and to see if the levels of OH-PAHs in the population decrease in line with the 3

70

decrease in PAH concentrations in ambient air and floor dust in residential houses, which was

71

observed over the last decade (Wang et al, 2016; 2019). Therefore, the aim of this study is to

72

measure the concentrations of OH-PAHs in pooled urine samples from a sample set of the

73

Australian population (stratified and pooled by age and sex) over two consecutive collection

74

periods of 2014-2015 and 2016-2017. Moreover, we also aim to provide the measurement of

75

cotinine, a biomarker of nicotine and the best available biomarker for tobacco smoke exposure,

76

for each pool to evaluate the contribution of smoking to the overall PAH exposure in this

77

population.

78 79

2. Materials and Methods

80

2.1 Collection of urine samples and pooling protocol

81

Similar to previous studies (e.g. Thai et al., 2016; He et al., 2018; Heffernan et al, 2015), we

82

utilised de-identified urine samples from surplus specimens that had been analysed for other

83

clinical pathology testing at a state-wide pathology laboratory in Queensland, Australia. Most of

84

the urine samples came from the South East Queensland region with a population of more than 3

85

million people including urban (Brisbane) and suburban areas. Urine samples were stored in

86

sterile polypropylene specimen containers together with descriptive information including date

87

of sample collection, the donor’s sex and birthdate. The surplus samples were gathered and

88

archived over time and subsequently pooled when sufficient number of samples were available.

89

As such the pooled samples are not only averaging the exposure in the population but also

90

averaging the exposure level over the year. For pooling purposes, individual samples were

91

stratified by age (calculated from the birthdate and the date of urine collection) and sex and then

92

pooled to six age strata (0-4, 5-14, 15-29, 30-44, 45-59 and ≥60 years) and two sex strata, with a

93

replicate for each strata.

94

For each sampling cycle (i.e. 2014-2015 and 2016-2017), a total of 2400 individual samples

95

were combined into 24 pools, with specimens from 100 individual samples in each pool,

4

96

representing two replicate pools for two sexes and six age strata. Because samples were pooled

97

based on equal volume (1 mL) from each individual sample, the concentration measured in each

98

pool is equivalent to the arithmetic mean of the concentration in each individual sample

99

contributing to the pool. Two sampling cycles were conducted to cover the period of 2014-2015

100

and 2016-2017. Consequenlty, 48 pools of urine from 2 sampling cycles were available for

101

analysis. This work was approved by the University of Queensland ethics committee (approval

102

number 2013000317).

103 104

2.2 Urine analysis

105

Analysis of OH-PAHs

106

Pooled urine samples were extracted and analysed for ten OH-PAHs using a modification of the

107

method described previously by Li et al. (2014) using gas chromatography-isotope dilution-

108

tandem mass spectrometry. The parent PAHs and the ten OH-PAHs analysed are presented in

109

Table 1. Briefly, urine samples (1 mL) were spiked with 13C-labelled internal standards (4 ng of

110

13

111

13

112

hydrolyse possible urinary conjugates of OH-PAHs overnight at 37 oC. The target OH-PAHs

113

were then extracted by liquid–liquid extraction twice with 5ml of 1:4 toluene:n-pentane. The

114

extracts were fortified with 50 µL of n-nonane as a keeper solvent before evaporated to near

115

dryness and transferred into a vial insert. Then, 0.5 ng of 13C12 PCB141 was added as a recovery

116

standard before the addition of 10µL of N,O-Bis(trimethylsilyl)trifluoroacetamide +

117

trimethylchlorosilane (BSTFA + TMCS ; 99:1). Air in the extract was displaced with nitrogen

118

and the samples incubated at 60ºC for 90 minutes.

119

Table 1. List of target OH-PAHs including abbreviations and parent chemicals

C6 1-NAP and 1 ng each of 13C6 3-fluoranthene (3-FLU),

13

C6 1-phenanthrene (1-PHEN) and

C6 1-pyrene (1-PYR)) and sodium acetate buffer containing β-glucuronidase (HP-2) enzyme to

Parent chemicals

OH-PAH names Carbaryl (for 1-NAP only) 1-naphthol Naphthalene 2-naphthol

Abbreviation 1-NAP 2-NAP

5

Fluorene

Phenanthrene

Pyrene

2-hydroxyfluorene

2-FLU

3-hydroxyfluorene

3-FLU

9-hydroxyfluorene

9-FLU

1-hydroxyphenanthrene

1-PHE

2-hydroxyphenanthrene

2- PHE

3-hydroxyphenanthrene

3- PHE

4-hydroxyphenanthrene

4- PHE

1-hydroxypyrene

1-PYR

120 121

GC separation was carried out using a DB-5MS column (30 m×0.25 mm i.d.; 0.25 µm film

122

thickness, J&W Scientific). The temperature program was set initially at 80 °C for 2 min and

123

then increased to 180 °C at 20 °C min−1, held for 0.5 min and increased further to 300 °C at 10

124

°C min−1 and held at this temperature for 5 min. The flow rate was maintained at 1.0 mL min−1.

125

The programmed temperature vaporization (PTV) injector temperature was held at 80 °C during

126

injection for 0.1 min, then increased to 200 °C at 14.5 °C s−1 and held for 1 min. One µL of

127

sample was injected, in splitless mode. Electron ionization (EI) mode was used and the

128

triplequad mass spectrometer was operated in the multiple reactions monitoring (MRM) mode

129

with an emission current of 20 µA. The transfer line and ionization source temperatures were set

130

at 280 °C and 270 °C, respectively. The collision gas pressure was 1.5 mTorr and the cycle time

131

was 0.4 s. Q1 peak width (FWHM) was set to 0.7 amu. MRM transitions, collision energy for

132

each transition, and average retention times (RTs) are presented in Table S1.

133

Four samples from our previous study (Thai et al. 2016) were re-run as part of this study and

134

used as quality control (QC) samples. In these four samples, all the target compounds were

135

detected. The newly measured concentration (ng/L urine) of each compound in those samples

136

was compared against the values reported previously. The accuracy was calculated as the values

137

derived from the current study against the one referred to. The reference values, QC values and

138

accuracy are presented in Table S2.

6

139

Concentrations of 1-NAP and 2-NAP of each pooled sample were used to calculate the 1-

140

NAP/2-NAP ratio. This ratio has been used as a parameter to evaluate whether the person or

141

group (in our case) was exposed to carbaryl or not (Meeker et al., 2007).

142 143

Analysis of cotinine

144

The biomarker of tobacco smoke exposure, cotinine, was measured in pooled urine samples by a

145

LC-MS/MS method using direct injection mode (Banks et al., 2018). A Phenomenex Kinetex

146

Biphenyl column (50 × 2 mm, 2.6 µm) kept at 45°C was used for separation. The flow rate was

147

0.3 mL/min. The mobile phase utilised a linear gradient from 5% B to 100% B in 6 minutes then

148

held at 100% for 4 minutes followed by equilibration at 5% B for 4 minutes (A = 0.1% formic

149

acid in MilliQ water, B = 0.1% formic acid in LCMS grade methanol). A Sciex 6500+ triplequad

150

mass spectrometer was operated in MRM mode. Samples were analysed in a batch with a blank

151

and QAQC injection every six to eight samples. Cotinine was quantified using the isotope

152

dilution method (using cotinine D3). LOD and LOQ were 17 ng/L and 51 ng/L, respectively.

153

The method was also validated for inter-and intra-day accuracy, precision, linearity and relative

154

matrix effect (Table S3).

155 156

2.3 Statistical Analysis

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Statistical analysis was performed using Microsoft Excel and GraphPad Prism (version 8.00,

158

GraphPad Software Inc.). The Shapiro-Wilk test indicated that concentrations of OH-PAHs and

159

cotinine in the pooled urine samples did not have a normal distribution. The Shapiro-Wilk test

160

was repeated once data had been log10 transformed, which indicated normal distribution of data.

161

Log10 transformed data were then used for statistical analysis. Differences in the mean between

162

sets of data were determined using the Student’s t-test. Bivariate correlations (Pearson

163

correlation coefficients) were used to investigate the correlations between the concentrations of

7

164

OH-PAHs and cotinine. Statistical significance was set at p < 0.05. For calculations, half the

165

method detection limit (LOD/2) was used when concentrations were below the LOD.

166 167

3. Results and Discussion

168

All OH-PAHs were detected in all of the samples. Cotinine was detected in 85% of the samples,

169

with all non-detectable pooled samples being the 0-4 age groups. There are three samples whose

170

internal standard recoveries were <50%, not satisfying the QAQC criteria. Consequently, their

171

OH-PAH concentrations were not presented or included in further data analysis. Individual

172

results of the 2014-2015 and 2016-2017 cycles are presented in Table S4 and Table S5,

173

respectively. The concentrations of OH-PAHs differ between individual OH-PAHs with

174

geometric means (GM) ranging from 40 ng/L (4-PHE) to 7800 ng/L (1-NAP). The overall GM

175

of 1-PYR, the most common biomarker for PAH exposure, were 100 and 120 ng/L in 2014-15

176

and 2016-17 sampling cycles, respectively.

177

In agreement with our previous results (Thai et al. 2016), the concentration of each metabolite

178

among age groups varied within one order of magnitude between the maximum to minimum

179

concentrations. An exception was 1-NAP, whose concentrations varied up to 200 folds in both

180

sampling cycles.

8

60

45 -5 9

30 -4 4

15 -2 9

514

04

Concentration (ng/L urine)

60

45 -5 9

30 -4 4

15 -2 9

514

04

Concentration (ng/L urine)

60

45 -5 9

30 -4 4

15 -2 9

514

04

Concentration (ng/L urine)

181 182

Fig. 1: Urinary concentration versus age of 1-NAP (ng/L) and 1-PYR (ng/L) for three sampling cycles (∆ - female pools;

183

line indicates mean concentration of each age strata. Note log axis for 1-NAP. Data of 2013 are from Thai et al. (2016).

- male pools). Horizontal

184 185 9

186

Table 2: Urinary concentrations of OH-PAHs (ng/L) in selected populations (geometric mean or median). Canada4 2014-15

Vietnam7 Australia7 2011-12 2011-12

Australia2 2016-17

24x100

24x100

24x100

2581

2487

2492

2640

2422

2511

2500

1016

1864

161

151

1-NAP

9221

5600

7800

2580

2050

1670

1520

1500

1000

970

820

6820

2451

953

2-NAP

4104

4500

7100

3830

3550

4140

4220

3800

4100

4600

1620

14350

2778

1456

1-NAP/ 2-NAP

2.2

1.2

1.1

0.7

0.6

0.4

0.4

0.4

0.2

0.2

0.5

0.5

0.9

0.7

2-FLU

261

350

250

303

240

240

181

270

260

280

n/a

3290

267

108

3-FLU

132

160

120

116

95

94

80

96

100

100

n/a

n/a

121

33

9-FLU

299

240

180

337

255

245

n/a

160

150

150

n/a

11810

614

124

1-PHE

134

-

-

139

131

126

93

150

150

160

n/a

2720

262

60

2- PHE

60

67

40

64

64

61

n/a

67

61

62

n/a

1930

114

30

3- PHE

81

-

-

98

72

62

n/a

87

83

89

n/a

3840

157

53

4- PHE

30

68

62

n/a

n/a

21

n/a

25

21

23

n/a

3530

n/a

n/a

1-PYR

142

100

120

118

119

111

132

110

88

96

27

8110

291

64

1

Canada4 Canada4 2011-12 2012-13

China6 Italy5 2013-14 2011-12

Australia2 2014-15

n

187

3 US 3 US 3 US 3 US 2007-08 2009-10 2011-12 2013-14

Australia1 2012-13

Thai et al. (2016); 2 This study; 3 CDC (2019); 4 Health Canada (2017); 5 Tombolini et al. (2018); 6 Sun et al. (2017): 7 Thai et al. (2015)

188 189 190 191

10

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3.1 Concentrations of OH-PAHs vs age/sex

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In general, concentrations of OH-PAHs measured in this study followed trends that were

194

observed in the previous sampling cycle (2013-2014), which means concentrations are higher in

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the adolescents and adult groups than in small children (0-4 years), school-aged children (5-14

196

years) and the elderly (>60 years) with some exceptions such as the high level of 1-NAP in the

197

elderly in 2015 and 2017 and the lower level of 1-PYR in the middle age group (30-44 yo) in

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2017 (Fig. 1). Profiles of the other seven OH-PAHs are presented in Fig. S1.

199

This study continues to deliver unique OH-PAH biomonitoring data for small children (0-4

200

years), who are often not included in biomonitoring programs due to difficulty in recruitment

201

and sample collection. It is encouraging to observe that concentrations of all OH-PAHs

202

measured in children of both preschool- (0-4 years) and school-aged (5-14 years) were

203

consistently lower than in adolescents and adults. These findings suggested lower exposure to

204

PAHs in children in Australia, which in turn protects this susceptible sub-population from

205

potential negative impacts due to exposure to high levels of PAHs (Perera et al. 2014). The mean

206

level of 1-PYR, the most commonly used PAH exposure biomarker, measured in children from

207

this study was lower than those reported for 3 year old children in Krakow, Poland (Sochacka-

208

Tatara et al., 2018), or school-aged children in Chongqing, China (Liu et al., 2012) but higher

209

than those measured in elementary school girls (6-8 years old) in Northen California, US

210

(Dobraca et al., 2018) and comparable to the levels reported for children in Germany (Becker et

211

al., 2007).

212

The observed relationship between OH-PAH concentration and age is not universal. While

213

biomonitoring in Canada (Canadian Health Measures Survey - CHMS) reported similar positive

214

association between OH-PAH concentrations and ages for all their monitoring cycles (Khoury et

215

al., 2018), the national biomonitoring program in the US (National Health and Nutrition

216

Examination Survey - NHANES) did not report such a trend (Bain, 2015; CDC, 2019). In fact,

217

age was negatively associated with the concentration of 1-PYR in the NHANES data set (Bain,

11

218

2015) although there are only three age groups in NHANES (6-11 years, 12-19 years, and > 20

219

years).

220

We observed no clear difference between urinary concentrations of OH-PAH and sex in general

221

although there were some exceptions (e.g. the case of 1-PYR concentrations between male and

222

females for the age group of 45-59 years in 2017 as shown in Fig. 1). This finding is consistent

223

with reports of previous studies from Israel, Spain and the US on urinary OH-PAHs (Levine et

224

al. 2015, Bartolomé et al. 2015; CDC, 2019). However, in Iran and Korea, males had

225

significantly higher OH-PAH concentrations in urine samples than females, mostly due to the

226

higher smoking rate in males in those countries (Sul et al. 2012; Hoseini et al., 2018).

227

The levels of OH-PAHs measured in this study, with the exception of 1-NAP, are comparable

228

with those reported for the US (CDC, 2019) and Canada (Health Canada, 2017) (Table 2). As

229

seen in Table 2, the level of urinary 1-PYR, the most common biomarker for PAH exposure, in

230

the Australian population are not only comparable with those reported for the US, Canada, but

231

also with Germany and Korea while considerably lower than those reported in developing

232

countries where the populations were regularly exposed to biomass fuel burning or traffic

233

pollution. In some studies, the concentrations of urinary 1-PYR can be as high as 1646 ng/L after

234

exposure to biomass burning (Hemat et al. 2012) or ~1000 ng/L after exposure to traffic

235

pollution (Wertheim et al. 2012).

236

It is noted that over the last decade, studies in Australia have reported a decrease in PAH

237

concentrations in ambient air and residential dust (Wang et al., 2016; 2019) but the results of this

238

study indicate that the level of urinary OH-PAHs were influenced more by the behaviours (e.g.,

239

tobacco smoke exposure) than the ambient air.

240 241

3.2 Relationship of OH-PAHs with biomarker of smoking

242

Smoking has been identified as a major source of PAH exposure in the general population

243

(Srogi, 2007), or even as the dominant factor influencing the urinary concentrations of OH12

244

PAHs of NHANES participants (Navarro et al., 2019). In this study, we measured concentrations

245

of urinary cotinine (the best biomarker of nicotine) to evaluate the influence of smoking on the

246

level of OH-PAHs. Cotinine concentrations of each pooled sample are presented in Table S4 and

247

Table S5 along with concentrations of OH-PAHs. The concentrations of cotinine were < LOD or

248

were very low in samples of the small children group (0-4 y) and school-aged children group (5-

249

14 y). In contrast, cotinine concentrations were much higher in adolescent and adult samples

250

(Fig. 2). This is supported by the significant correlation between cotinine concentrations with all

251

metabolites of fluorene, the main PAH in cigarette smoke (Ding et al., 2005) as shown in Table

252

S6. This similarity demonstrates that smoking is an important factor contributing to the age-

253

dependent profile of OH-PAH concentrations in Australia similar to the NHANES program

254

(Navarro et al., 2019).

255

Cotinine concentrations in samples of small children (0-4 years) were all
256

exposure to cigarette smoke. In samples of school-aged children (5-14 years), cotinine

257

concentrations were low with the maximum concentration of 36 µg/L. Although this level of

258

cotinine was low, the presence of cotinine in samples of school-aged children (5-14) indicated

259

the influence of second hand smoking and perhaps underage smoking. Cotinine concentrations

260

increased markedly in adolescents and adults, which fits well with the fact that smoking rates are

261

higher among adolescents and adults than in children and the elderly (ABS, 2019). In Fig. 2a, it

262

could be observed that the influence of smoking (first hand or second hand) are different

263

between males and females in the age groups of 15-29 and 30-44 and less clear in the 45-59 and

264

>60 age groups, which was in agreement with smoking trends reported by the Australian Health

265

Survey as well (ABS, 2019).

13

266

Fig. 2: Urinary concentration of cotinine (ng/L) a) versus age (years) and b) between males and

267

females (∆ - female pools;

268

Interestingly, a t test showed no significant difference between the levels of cotinine in males

269

and females in the adult groups (Fig. 2b). It is likely that the difference in smoking prevalence

270

among males (16.9% and 16.5% in 2014-15 and 2017-18, respectively) and females (12.1% and

271

11.1% in 2014-15 and 2017-18, respectively) in Australia are small (ABS, 2019) and the sample

272

size is small to assess the significance of differences in cotinine concentrations by sex, although

273

a difference can be observed visually in some age groups (Fig. 2b).

- male pools).

274 275

3.3 Urinary concentrations of 1-NAP and 2-NAP in the studied population

276

Among the OH-PAHs measured in this study, 1-NAP is known as not only the

277

metabolite/biomarker of naphthalene but also of carbaryl (1-naphthyl-N-methylcarbamate), a

278

broad-spectrum carbamate insecticide. Meanwhile 2-NAP only arises from exposure to

279

naphthalene.

280

Concentrations of 1-NAP measured in this study varied much more widely than the other OH-

281

PAHs, with the difference between maximum and minimum values of about 200 fold in both

282

years (Table S4 and Table S5). Meanwhile, concentrations of 2-NAP varied within one order of

283

magnitude. Three samples in the 2014-15 cycle and eight samples in the 2016-17 cycle have

284

concentrations of 1-NAP > 20000 ng/L, a value near the 95th percentile of 1-NAP concentrations 14

285

reported in the US NHANES (CDC, 2019) and above the 95th percentile of 1-NAP

286

concentrations reported in the Canadian CHMS (Health Canada, 2017). The presence of some

287

pooled samples with very high concentrations of 1-NAP has resulted in higher average

288

concentrations of 1-NAP in Australia compared to the levels reported in the US or Canada

289

(Table 2). Average concentrations of 2-NAP were mostly comparable with data from other

290

countries except for the average concentration of the last sampling cycle in 2016-2017.

291

The consequence of the elevated level of 1-NAP is that, as we can see in Table 2, the ratio of 1-

292

NAP/2-NAP in Australia is > 1 while it is < 1 in all the other countries listed. Meeker et al.

293

(2007) have suggested that a higher ratio of 1-NAP/2-NAP may indicate exposure to carbaryl in

294

addition to naphthalene. They have also proposed a ratio of 1-NAP/2-NAP >2 as a threshold to

295

identify the contribution of carbaryl exposure to the urinary concentration of 1-NAP.

296

In this study, five samples in the 2014-15 cycle (25% of the samples) and nine samples in the

297

2016-17 cycle (38% of the samples) had the 1-NAP/2-NAP ratios >2, ranging from 2.1 to 35,

298

which were a very high proportion . All 14 samples of high 1-NAP/2-NAP ratio belong to adult

299

age groups from >30 years old, which is consistent with our previous report (Thai et al., 2016).

300

The issue of contamination during sample handling is highly unlikely because i) none of the

301

samples from infant and children groups in all three cycles had 1-NAP/2-NAP ratio > 2 and ii)

302

none of the samples (312) of the study by Thai et al. (2015), which were handled by a similar

303

protocol had 1-NAP/2-NAP ratio > 2. The two compounds, 1-NAP and 2-NAP, are relatively

304

stable in urine, with 1-NAP slightly less stable than 2-NAP (Lee et al., 2008; Gaudreau et al.,

305

2016). Therefore, it is unlikely that 2-NAP would degrade faster than 1-NAP in urine to give a

306

higher 1-NAP/2-NAP ratio.

15

307

Fig. 3: Urinary concentration of 1-NAP and 2-NAP (ng/L) in children and adult samples. Data

308

of 2013 are from Thai et al. (2016).

309

While the level of 1-NAP in children has been stable over the 3 sampling cycles, the level of 2-

310

NAP in the same age groups has gradually increased (Fig. 3b). It is difficult to explain the reason

311

of such increase in 2-NAP and 4-Phe (Fig. S2) in children while the level of 1-NAP and other

312

OH-PAHs measured in this study were relatively stable. Jung et al. (2014) observed a similar

313

phenomenon in children in New York (decrease of 1-NAP but increase of 2-NAP) and the

314

authors suggested it was due to the reduction in exposure to carbaryl and increased exposure to

315

outdoor sources. It could well be the case but at the same time, it is intriguing to observe the

316

reverse phenomenon where 1-NAP/2-NAP ratio was <0.5 (or 2-NAP/1-NAP >2), both in this

317

study and in Jung et al. (2014), especially when the level of 1-NAP was similar in both studies.

318

Children from Krakow, Poland also exhibited a low 1-NAP/2-NAP ratio (<0.5) with a mean 1-

319

NAP value of 1916 ng/L (Sochacka-Tatara et al., 2018).

320 321

3.4 Carbaryl or not carbaryl?

322

In Meeker et al. (2007), which is often cited in biomonitoring studies measuring 1-NAP and 2-

323

NAP, a ratio of 1-NAP/2-NAP > 2 is suggested as a threshold indicating exposure to carbaryl.

16

324

According to this criteria, many people in the population monitored by our study could have

325

been exposed to carbaryl.

326

However, in our opinion, it is unlikely that Australians in this population were exposed to

327

carbaryl at higher frequency than other countries like the US or Canada as shown by the data

328

shown in Table 2, especially when the use of carbaryl as insecticide in domestic situations in

329

Australia is more restricted after a review of the Australian Pesticides and Veterinary Medicines

330

Authority (APVMA) in 2007, with many products having their registrations cancelled (APVMA,

331

2014).

332

Although the urinary concentration of 1-NAP after exposure to carbaryl could be very high and

333

could have ‘contaminated’ the urine samples of our study, it should also be noted that the

334

inclusion of 100 samples per pool allows for considerable dilution. To increase the 1-NAP

335

concentration of the pooled samples from the overall mean of ~6000-9000 ng/L to > 20000 ng/L,

336

a value near the 95th percentile of NHANES study found in several samples of this study, the

337

contaminated individual specimen would have a 1-NAP concentration of approximately

338

2,000,000 ng/L, similar to the level found in urine in farmers after carbaryl application as

339

reported by Shealy et al. (1997). The maximum 1-NAP concentration measured in urine of a

340

participant after receiving a maximum allowable daily dose of carbaryl was only 200 µmol/mol

341

creatinine or approximately 300,000 ng/L was (Sams, 2017), which would contribute much less

342

of an increase to the concentration of the pooled samples.

343

The high frequency of finding a 1-NAP/2-NAP ratio that is very high in urine samples in the

344

literature as presented below makes the case of carbaryl exposure more unlikely. In a Chinese

345

cohort study, Zhang et al. (2014) reported 1-NAP/2-NAP ratio in the range of 10-20. In that

346

study, the average 1-NAP concentration in participants was >50000 ng/L after consumption of

347

various food while the average concentrations of 2-NAP were 5000 ng/L and 2000 ng/L in

348

female and male participants, respectively. In another study, high ratios of 1-NAP/2-NAP were

349

also reported in a controlled dietary exposure study involving nine persons consuming barbecued 17

350

chicken (Li et al., 2012). The authors attributed the higher than expected 1-NAP/2-NAP ratios

351

(91-442) in three participants to exposure to carbaryl, which is unlikely in a controlled

352

biomonitoring study such as the case of Li et al. (2012). In another study, the concentration of 1-

353

NAP in a Standard Reference Material for urinary OH-PAHs in non-smokers was found to be

354

140 times higher than the GM value for US non-smokers, leading to a 1-NAP/2-NAP ratio of

355

>150 (Schantz et al. 2015). Carbaryl was also suggested as contributing to the high urinary

356

concentrations of 1-NAP.

357

Again, in our opinion, the attribution to carbaryl exposure alone is not sufficient enough to

358

explain the occurrence of many cases as decribed above, especially the consistent occurrence of

359

high 1-NAP/2-NAP ratio (>2) in several pooled samples in Australia over a period of six years.

360

Other source(s) or cause(s) should be explored and investigated.

361

Our first hypothesis is that there are other sources contributing to the presence of 1-NAP in

362

human urine samples. One of these sources are cosmetic products, specifically in hair dyes (King

363

et al., 2018). After the application of hair dyes, 1-NAP can be found in urine in both free and

364

glucuronide forms (SCCNFP, 2001) although the actual impact of 1-NAP in those products that

365

can have on the level of 1-NAP in urine has not been properly assessed (SKHBU, 2007).

366

Our second hypothesis is that the rate of metabolism of naphthalene varies with the level of

367

exposure to naphthalene, which was also suggested in a recent review by de Oliveira et al.

368

(2014). It can be seen in Table 2 that in the surveys of the general population of Canada, Italy

369

and the US, the ratio of 1-NAP/2-NAP is < 1, averaging 0.5. It means that metabolism of

370

naphthalene is skewed toward 2-NAP at low level of exposure. When people are constantly

371

exposed to higher level of naphthalene, more 1-NAP would be generated, increasing the 1-

372

NAP/2-NAP ratio. Thai et al. (2015) have found that the ratio of 1-NAP/2-NAP has significantly

373

increased when the participants travelled from Brisbane (low exposure) to Hanoi (high

374

exposure). A similar finding was reported for travellers from Los Angeles to Beijing by Lin et al.

18

375

(2016). Furthermore, studies into occupational exposure to naphthalene also reported ratios of 1-

376

NAP/2-NAP at ~2 or >2 (Klotz et al., 2019; Sams, 2017; Serdar et al., 2003).

377

Although this study lacks concrete evidence to dismiss the proposition of carbaryl exposure in

378

the studied population, the results warrant further investigation into other sources of 1-NAP or

379

different metabolic mechanisms that can skew the ratio of 1-NAP/2-NAP in human urine.

380 381

3.5 Limitations

382

The use of samples of convenience from a pathology laboratory could affect the

383

representativeness of the metabolism to chemicals in the general population. The use of pooled

384

samples prevented the detection of persons having extremely high or low concentrations or the

385

inter-individual variations in the population. A discussion of the opportunities and limitations of

386

using pooled samples for biomonitoring can be seen elsewhere (Heffernan et al., 2014).

387 388 389

4. Conclusions

390

This study continues to provide the information on PAH exposure of an Australian population

391

including small children. According to our data, the level of exposure to PAHs (3-4 rings) in

392

Australia were relatively low compared to other countries in the world. However, the urinary

393

concentration of 1-NAP (and consequently the ratio of 1-NAP/2-NAP) are relatively high in

394

some samples. We proposed two explanations: i) other exposure sources including carbaryl and

395

1-NAP itself in cosmetic products; and ii) different rates of naphthalene metabolism at different

396

levels of exposure to naphthalene. Further study is needed to validate these explanations.

397

In general, our results showed that children and the elderly are less exposed to many PAHs than

398

adolescent and adult groups (15-49 years), with a considerable influence from exposure to

399

cigarette smoke in the latter groups as reflected by their cotinine concentration profiles.

400 19

401

Acknowledgments

402

The Queensland Alliance for Environmental Health Sciences, The University of Queensland

403

gratefully acknowledges the financial support of the Queensland Department of Health. The

404

authors wish to thank Dr Soumini Vijayasarathy and the staff at Sullivan Nicolaides Pathology

405

Taringa for assistance with sample preparation. JFM is funded by an UQ Fellowship. PT was

406

partly funded by an ARC Discovery project (DP180101475). The authors would like to thank

407

the Australian Government Department of the Environment for their financial support. Dr. Sara

408

Broomhall is gratefully acknowledged for ongoing discussion and assistance to the QAEHS

409

researchers. The authors declare no conflict of interest.

410 411

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25

Highlights - OH-PAHs in an Australian population were monitored using pooled urine samples. - Samples pooled from 4800 individual over two cycles in 2014-15 and 2016-17 were analysed. - Cotinine concentrations were much higher in samples from adolescences and adults than from children - Elevated level of 1-naphthol was measured in some adult urine samples over the period. - High 1-naphthol concentrations potentially due to unrecognized exposure sources and/or differential metabolism.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: