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Food Safety and Toxicology

Effect of propiconazole on the lipid metabolism of zebrafish embryos (Danio rerio) Miaomiao Teng, Feng Zhao, Yimeng Zhou, Sen Yan, Sinuo Tian, Jin Yan, Zhiyuan Meng, Sheng Bi, and Chengju Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00449 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 7, 2019

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Journal of Agricultural and Food Chemistry

Effect of propiconazole on the lipid metabolism of zebrafish embryos (Danio rerio)

Miaomiao Teng1, Feng Zhao1, Yimeng Zhou1, Sen Yan2, Sinuo Tian2, Jin Yan2, Zhiyuan Meng2, Sheng Bi3, Chengju Wang1*

1. Department of Applied Chemistry, College of Science, China Agricultural University, Beijng, China 2. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Science, China Agricultural University, Beijng, China 3. Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Correspondence author Chengju Wang Tel: +86(0)10-62733924 Fax: 010-62734294 E-mail: [email protected] Address: Yuanmingyuan West Road 2, China Agricultural University, Beijing, 100193, People’s Republic of China

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Abstract

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Propiconazole is a triazole fungicide that has been widely used in agriculture and has

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been detected in the aquatic environment. This study aimed to investigate the effects of

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propiconazole exposure on lipid metabolism in the early life stages of zebrafish for 120

5

hours post-fertilization (hpf). Using the early life stages of zebrafish to address

6

scientific questions is lower cost, more efficient and suitable for meeting current

7

legislation than other traditional fish species. Exposure to propiconazole significantly

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inhibited the development of zebrafish embryos and larvae. This exposure also caused

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reduced locomotor activities in zebrafish. Furthermore, total cholesterol levels,

10

lipoprotein lipase and fatty acid synthase activities were significantly decreased. The

11

expression levels of genes involved in lipid metabolism were significantly up-regulated

12

in response to propiconazole exposure. GC-MS/MS analysis revealed that fatty acids

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were significantly decreased. Together, the findings indicate the potential

14

environmental risk of propiconazole exposure in the aquatic ecosystem.

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Keywords: propiconazole, zebrafish embryo, lipid metabolism, fatty acids

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Introduction

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Propiconazole (PCZ) (1-((2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl)methyl-

18

1H-1,2,4-trizole) is a broad-spectrum and high efficiency triazole fungicide that has

19

been widely used in agriculture and horticulture to inhibit fungal growth

20

propiconazole is a common fungicide used for crop farming and is easily transported to

21

the ecosystem through spray drift, surface run-off, and rainfall, its residues have been

22

detected in the aquatic environment (Table 1), as well as in vegetables, fruits, and

23

human sera

24

environment has a potentially adverse effect on some aquatic organisms such as

25

Daphnia manga and Channa punctata Bloch 7, 8.

26

Previous reports have also shown that propiconazole can cause toxic effects on

27

vertebrates. Levels of the reactive oxygen species (ROS) were increased in cultured

28

hepatic cells and in mouse liver after exposure to propiconazole, and the changes

29

resulting from propiconazole were also revealed in mice via genomics and proteomics

30

9, 10.

31

induces hepatocarcinogenesis of mice 11. Juvenile rainbow trout (Oncorhynchus mykiss)

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exposed to propiconazole exhibit various physiological responses, including hepatic

33

ethoxyresorufin-O-deethylase (EROD) activity, antioxidant indices, morphological

34

indices, and hematological parameters

35

activity of lanosterol-14α-demethylase enzyme, the one that is essential for ergo sterol

36

biosynthesis, and suppresses cytochrome P450 enzyme activity (CYP51) to block

37

fungal cell wall chitin

3-6.

1, 2.

Since

Recent studies have reported that propiconazole in the water

In addition, data have shown that propiconazole affects hepatic metabolism and

13.

12.

Additionally, propiconazole inhibits the

Triazole fungicides have been considered mainly to affect 3 ACS Paragon Plus Environment

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lipid biosynthesis and metabolism pathways 14-17.

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Lipids play an important role in energy supply and maintaining normal metabolic

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activities 18. Lipid homeostasis requires precise control of lipid process, including lipid

41

accumulation, lipogenesis, fatty acid β-oxidation, cholesterol synthesis and metabolism.

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Disordered homeostasis leads to obesity, malnutrition, endocrine disruption, or other

43

metabolic-associated diseases

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syndrome in 21 century. The WHO has reported that there are at least 41 million

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children under 5 years old with obesity or overweight 23. One possibility is that a rise

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in environmental pollutants may contribute to obesity or other health problems

47

Thus, the investigation of the effects of environmental chemical exposure on the lipid

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metabolism of organisms in the early stages is urgent. Data have shown seasonal

49

variation in lipid metabolism of yellow perch (Perca flavescens) chronically exposed

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to sub-lethal levels of heavy metals (Cd, Zn, Cu) (Rouyn-Noranda, Quebec)26. Studies

51

have also shown that nano-sized particles transported through the food chain affect

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behavior and lipid metabolism in Crucian carp (genus Carassius), Bleak (Alburnus

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alburnus), Rudd (Scardinius erythrophthalmus), Tench (Tinca tinca), Pike (Esox esox),

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and Atlantic salmon (Salmo salar)27. Carnevali., et al have reported that the mixtures

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of endocrine disruption chemicals (EDCs) affect lipogenesis and fat deposition in the

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Juvenile seabream 28. Different types of toxicant substances alter lipid metabolism in

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marine and fresh water fish. Additionally, previous studies demonstrate that

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propiconazole increases cell proliferation and Ras farnesylation in AML12 mouse

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hepatocytes via the dysregulation of the cholesterol biosynthesis pathway 29. Therefore,

19-22.

For instance, obesity is an epidemic metabolic

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it is important to investigate the potential mechanism of the effect of propiconazole

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exposure on lipid metabolism in the early stages of freshwater organisms.

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The zebrafish embryo, as an ideal aquatic vertebrate model, has a rapid life cycle, and

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is easy to observe its morphological process during the development because the

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embryo is transparent

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questions is more suitable for meeting current legislation

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genes in mammals have been identified in zebrafish

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used for investigating the toxic effects of environmental pollutants 17, 34, 35.

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In the present study, we investigated the developmental effects, locomotor activity,

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lipid synthesis and metabolism change, and free fatty acid alterations in the early stages

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of zebrafish with exposure to different concentrations of propiconazole (0, 0.5 mg/L,

71

2.5 mg/, 4.5 mg/L). These results provide the insight into the underlying mechanism of

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the effects of propiconazole exposure on lipid metabolism, predicting the potential

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environmental risk of propiconazole to aquatic organisms.

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Material and methods

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Reagents. Propiconazole (CAS#: 60207-90-1; 95% purity) was obtained from China

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Ministry of Agriculture. The stock solutions were prepared in acetone (purity > 99%).

77

Standard water was used in the lab, containing 0.5 mM Mg2+, 2 mM Ca2+, 0.074 mM

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K+ and 0.75 mM Na+. 36. All other chemicals were at the analytical grade.

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Experimental design and sample collection. Five-month old wild-type zebrafish (AB

80

strain, Danio rerio) were obtained from Hongdagaofeng fish shop. The parental

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zebrafish were cultured in flow-through equipment (Esen Corp, Beijing, China) under

30.

In addition, using zebrafish embryos to address scientific

32, 33.

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Furthermore, most of

Therefore, the embryo was

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14h:10h light/dark at 28 oC for 14 days. Zebrafish were fed with Artemia nauplii twice

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daily. Zebrafish embryos were maintained according to our previous study 17. A range

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of concentrations (0, 10, 11, 13.2 14.52, 15.97, 17.57 mg/L and 0, 0.42, 0.67, 1.07, 1.72,

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2.75 mg/L) of propiconazole exposed to zebrafish embryos on the basis of pre-

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experiment data to test acute toxicity of embryo. Six replicates were used for each test

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solution of propiconazole (0, 0.5, 2.5, and 4.5 mg/L) based on the results from pilot

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studies. Control and propiconazole treatments received 0.01% (v/v) acetone. Two hours

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post fertilization (hpf), normal embryos were randomly assigned into the test groups.

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Each replicate contained 200 embryos in 500 mL solution. During the exposure period,

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the test solution was kept at 28 oC and the day/night cycle was 14h/10h. The exposure

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media was changed daily. The embryos from each group were observed and recorded

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using light microscopy at 24, 48, 72, 96, and 120 hpf. After 120 h exposure, zebrafish

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larvae were collected and frozen in liquid nitrogen, and then stored at -80 oC for further

95

analysis.

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Embryonic developmental test. During the propiconazole exposure, 10 larvae were

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selected as one replicate (n = 6 replicates) and put into 24-well plates to observe the

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embryonic development. We observed the spontaneous movement of embryos in 20s

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at 24 hpf and counted the heartbeat number in 20s at 48 hpf. The hatching rate was

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recorded at 72, 96, and 120 hpf. After 120 hpf, we recorded teratogenic effects and

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measured the body length of hatched individual larvae using digital microscope (Aigo

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GE-5, Beijing, China).

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Determination of larval locomotor activity. Based on the previous study, the 6 ACS Paragon Plus Environment

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locomotor activity of 120 hpf larvae was measured using the Video-Track system (UI-

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3240CP-C-GL, IDS Imaging Development System GmbH, Obersulm, Germany)

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following the manufacture’s protocol37. Average velocity, distance movement, active

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and inactive time, average acceleration and deceleration were recorded from 10 larvae

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per concentration every 10 mins (n = 6 replicates) and further analyzed using uEye

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Cockpit software Loligo System, United Ststes).

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Measurement of triglyceride (TG) and total cholesterol (TCHO) content. 30 larvae

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(n = 6 replicates) were homogenized in 270 μL cell lysates (Applygen, Beijing, China)

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and centrifuged at 3000 g for 10 min at 4 oC. Thus the supernatant was used for

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determinations of total protein using a bicinchoninic acid (BCA) protein assay kit

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(Cwbiotech, Beijing, China)16. And then the remained samples were incubated for 10

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min in 70 oC water to detect TG and TCHO contents according to the enzymatic kits

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(Applygen, Beijing, China) according to previous studies16. TG and TCHO were

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normalized to the sample protein concentration. The content of was measured

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Absorbance of TCHO, TG and total protein were measured using the microplate reader

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(Multiscan MK3, Thermo Scientific) at 540, 540, 595 nm, respectively. The calibration

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of protein was shown in Figure S1.

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Lipoprotein lipase (LPL) and fatty acid synthase (FASN) activity assay.

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Lipoprotein lipase and fatty acid synthase were extracted from 120 hpf larvae samples

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(30 larvae, n = 6 replicates). Samples were homogenized in 270 μL saline and

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centrifuged at 3000 g for 10 min at 4 oC. The supernatant was used to enzymatic activity

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analysis. The LPL and FASN activity assays were determined using enzyme-linked 7 ACS Paragon Plus Environment

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immunosorbent assay (ELISA) kits (Fu life Industry Co., Ltd. Shanghai, China)38. The

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range suitability of LPL and FASN were 1.6-65 U/mL, 10-360 U/L, respectively. The

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standard calibration of LPL and FASN were shown in Figure S2.

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Quantitative real-time polymerase chain reaction (qRT-PCR) assay. After 120 h

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exposure, 30 embryos were collected and dissolved in Trizol reagent for total RNA

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extraction according to previous method

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using FastQuant RTase kit (Tiangen Biotech, Beijing, China). Quantitative real-time

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polymerase chain reaction (qRT-PCR) was conducted using SYBR Green PCR Master

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Mix reagent kit (Tiangen Biotech) and performed using an ABI 7500 PCR system

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(Advanced Biosystems, Foster City, CA, USA). Thermal cycling was set at 95 °C for

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15 min, followed by 40 cycles at 95 °C for 10 s, 60°C for 20s, and 72°C for 32s.

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Transcription of target genes was calculated using the 2−ΔΔCt method. β-actin was

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chosen as the house-keeping gene. All primers of target genes were designed using

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Primer 6.0 software and synthesized by Sangon Biotechology (Shanghai, China) (Table

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S1).

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Metabolomics analysis based Gas chromatography-mass spectrometry (GC-MS).

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Embryos (80 embryo, n = 6 replicates) were extracted as previously described

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Briefly, 80 embryos were collected and homogenized in 20 μL internal standard (50

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μg/L C15:0 fatty acid and C17:0 methyl ester) and 600 μL extracting solution

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(chloroform : methanol = 1:2, v/v). Then, the homogenization was added into 200 μL

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chloroform and 200 μL water, vortexed for 2 min, and centrifuged at 18001 g for 10

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min. Extraction was repeated again and evaporated to dryness under a stream of

22 39

The first-strand cDNA was synthesized

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nitrogen, following derivatization with 1 m L methanol/hydrochloric acid (41.5/9.7 mL)

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for 12 h at 60 oC. The solvent was cleaned up by hexane-saline (1:1). After extracts

150

were evaporated by nitrogen, each solvent was dissolved with 200 μL hexane and then

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transferred to an injection vial for analysis. Free fatty acids were measured using a

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Thermo Fisher Scientific Trace GC gas chromatograph coupled to a Quantum XLS

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mass spectrometer (Thermo Scientific, Waltham, MA, USA) using a DB-5 column (30

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m×0.31 mm×0.25 mm), running in full scan mode. Data analysis was conducted with

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Thermo Xcalibur software. Fatty acids were quantified by normalizing the integrated

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peak areas to the internal standards.

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Propiconazole in water analysis

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Water samples were collected at the beginning of exposure (0 h) and at 24 h. All

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exposure solutions were analyzed for all treatments. The experimental solutions were

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filtered with a 0.22 μm filtration membrane and then determined using ultra-high

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performance liqud chromatography – tandem mass spectrometry (UHPLC-MS/MS)

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(ultiMate 3000 system,

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described 42, 43.

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Statistical analysis. All statistical analyses were performed with SPSS 19.0 (IBM,

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USA). Significant differences were determined by one-way ANOVA analysis on the

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basis of Dunnett post hoc comparison (P < 0.05). All values were presented as the mean

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± standard deviation (SD).

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Results

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Assay validation

Thermo Scientific, Waltham, MA, USA) as previously

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Spiked samples with 1, 50, and 2500 μ,/L of propiconazole were analyzed. The

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recovery of propiconazole in solutions ranged from 93.8% to 106.9%. (Table S2). An

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external standard calibration curve was used to calculate the amount of propiconazole

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(Figure S3). The range of propiconazole concentrations and linear regression equations

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was 1-1000 μg/L, R2=0.99, respectively. Concentrations of propiconazole in water (0,

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0.5, 2.5, 4.5mg/L) were detected. The measured propiconazole concentrations in water

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samples were 80-120% of nominal values (Table S3).

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Lethal effect of propiconazole

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According to the results, the 96 h half dose/lethal concentration (LD50/LC50) value of

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embryo (95% conference limit) was 12.90 (12.40-13.40) mg/L with linear equation Y=

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14.33X-15.92 (R2 = 0.97). The low observed effect concentration (LOEC) was 1.72

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mg/L and the no observed effect concentration (NOEC) was 1.07 mg/L at 96 hpf

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(Figure S4). There is no significance in other parameters.

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Toxicological endpoints in embryos.

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The effects of propiconazole exposure on the spontaneous movement, heartbeat,

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hatching rate, and body length of embryos and larvae were shown in Figure 1.

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Propiconazole exposure resulted in significant increases in the spontaneous movement

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at 24 hpf and significant decreases in the number of heartbeat at 48 hpf in the 2.5 mg/L

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and 4.5 mg/L propiconazole groups. Compared with the control group, the hatching

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rate was inhibited at 72, 96, and 120 hpf by 4.5 mg/L propiconazole exposure, but was

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not affected by other doses. We also observed reductions of body length in larvae

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exposed to 2.5 and 4.5 mg/L propiconazole for 120 h. Larvae with malformation 10 ACS Paragon Plus Environment

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following exposure to propiconazole at 120 hpf were shown in Figure 2. In the

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propiconazole-exposed groups, the spine deformation (Sd) and tail malformation (Tm)

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were observed, indicating that propiconazole induced the developmental toxicity of

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zebrafish embryos. Although the malformation rate was observed in the groups treated

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with high doses, there were no statistical significances (Figure 2H).

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Locomotor activity analysis.

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Locomotor activity was conducted in 120 hpf larvae (Figure 3). Under the light, average

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velocity and moving distance were significantly decreased in 2.5 and 4.5 mg/L

200

propiconazole groups. As the exposure concentrations increased, active times of larvae

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were significantly increased and inactive times were significantly decreased. Therefore,

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compared with the control group, the average acceleration was significantly decreased

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and the average deceleration was significantly increased in a dose-dependent manner.

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TG and TCHO contents.

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The effects of propiconazole exposure on TG and TCHO contents in larvae were shown

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in Figure 4. 120 h exposure to 2.5 mg/L propiconazole significantly reduced the levels

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of TCHO, while no significant changes of TG contents were observed in larvae exposed

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to propiconazole.

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LPL and FASN activity.

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After 120 h exposure, LPL enzyme activity of zebrafish larvae was significantly

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decreased in the 2.5 and 4.5 mg/L propiconazole groups (Figure 5A). The FASN

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activity was also significantly decreased in the larvae following propiconazole exposure

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(Figure 5B). 11 ACS Paragon Plus Environment

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Gene expression analysis.

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To further identify the effects of propiconazole on lipid metabolism in larvae, the

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transcription levels of genes associated with lipid metabolism were determined in the

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larvae with propiconazole exposure (Figure 6). As described in previous literatures,

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these genes are involved in lipid accumulation, lipogenesis, fatty acid (FA) β-oxidation,

219

and cholesterol metabolism 16, 33, 44. At 120 hpf, compared with the control group, the

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transcription

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palmitoyltransferase 1 (cpt1), and hydroxymethyl glutaryl coenzyme A reductase b

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(HMGCRb) were significantly increased in larvae exposed to 0.5 and 4.5 mg/L

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propiconazole.

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acyltransferase (apgat4), carbohydrate response element binding protein (chrebp),

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acetyl-CoA carboxylase 1(acc1), peroxisome proliferator-activated receptor-α (pparα),

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sterol 14α-demethylase – cytochrome P51(CYP51), and cytochrome P7A1 (CYP7A1)

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were significantly upregulated in all three groups of larvae after propiconazole exposure.

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The expression levels of sterol regulatory element-binding protein 1(screbf1), acyl-

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CoA oxidase 1 (acox1), and hydroxymethyl glutaryl coenzyme A reductase a

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(HMGCRa) were significantly upregulated in the 4.5 mg/L propiconazole group.

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Upregulation of 7-dehydrocholesterol reductase (DHCR7) was also observed in the 2.5

232

mg/L propiconazole group.

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Fatty acids analysis

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We further semi-quantitatively analyzed the composition of free fatty acids in larvae

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(Figure 7 and Table S4). The typical curve of a control (A) and 4.5 mg/L propiconazole

levels

The

of

diglyceride

transcription

levels

acyltransferase

of

(dgat2),

carnitine

1-acylglycerol-3-phosphate

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treatment (B) were showed in Figure S4. Compared with the control group, most of

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saturated fatty acids (C16:0, C18:0, C20:0, Figure 7A) and monounsaturated fatty acids

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(C20:1N9, C20:1n11, Figure 7B) were significantly decreased in zebrafish following

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propiconazole exposure for 120 hpf. In contrast, the content of C18:1N9 was increased

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in 2.5, and 4.5 mg/l propiconazole-treated groups. We did not detect polyunsaturated

241

fatty acids.

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Discussion

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In the present study, we investigated the developmental toxicity of the fungicide

244

propiconazole and its particular effects on lipid metabolism in the early life stages of

245

zebrafish. Propiconazole exposure significantly inhibited the development of zebrafish

246

embryos and larvae, showing lowered heartbeat, hatching rate, and body length and

247

decreased locomotor activities. Analysis of lipid metabolism revealed that

248

propiconazole exposure resulted in decreased activity of LPL and FASN.

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Propiconazole exposure also altered the expression of genes related to lipid

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accumulation, lipogenesis, fatty acids β-oxidation, as well as cholesterol synthesis and

251

metabolism. Together, these results indicate that propiconazole has developmental

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toxic effects likely via affecting lipid metabolism.

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Total cholesterol, as a multifunctional molecule, is an important constituent of lipid

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related to lipid raft45. In addition, TCHO was the tetracyclic hydrocarbon chemicals to

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be essential for the component of cell members and signal transduction between cells46.

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HMGCR plays a crucial role in cholesterol synthesis and is a limiting step of cholesterol

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biosynthesis

47.

CYP51, as the objective of triazole fungicides, catalyzes lanosterol 13 ACS Paragon Plus Environment

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demethylation in the process of cholesterol synthesis 16. The DHCR7 gene is the last

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step in cholesterol synthesis, which catalyzes the conversion of 7-dehydrocholesterol

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into cholesterol 48. In our study, the mRNA expression levels of four critical cholesterol

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synthesis transcripts (HMGCRa, HMGCRb, CYP51, and DHCR7) were significantly

262

up-regulated, leading to increased content of TCHO. In contrast, CYP7A1, as a key

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rate-limiting enzyme gene, plays an important role in maintaining the homeostasis of

264

lipids and participates in the cholesterol metabolism, through which process bile acids

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are generated

266

resulted in significantly decreased levels of TCHO following exposure to propiconazole,

267

suggesting that propiconazole affects lipid metabolism in the zebrafish larvae. Similarly,

268

the expression levels of HMGCRa, HMGCRb, CYP51, CYP7A1, and DHCR7 were

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significantly increased in female zebrafish exposed to triazole fungicide difenoconazole

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at environmentally relative concentrations for 15 days influenced the content of

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TCHO16. Skolness et al. also demonstrated that the reduction of TCHO content was

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found in the fathead minnow (Pimephales promelas) after three weeks exposure of

273

propiconazole

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TCHO and affected lipid metabolism in adult zebrafish liver, due to the lack of energy

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supply 51. It would be of interest to examine any alterations in glucose metabolism in

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zebrafish following propiconazole exposure.

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FASN, as a multifunctional single-chain protein, catalyzes saturated fatty acids

278

synthesis in cells 52, 53. Palmitate, a 16-carbon long-chain fatty acid (C16:0), is the major

279

product of fasn, which can undergo elongation by fasn Ⅲ-forming stearate (C18:0) 54.

49.

50.

Comprehensively, the alterations of genes related to cholesterol

Young et al. reported that thifluzamide caused decreased levels of

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Acox1 encodes a key lipogenic enzyme that catalyzes the conversion of acetyl-CoA to

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malnoyl-CoA and further converted by FASN to fatty acids 55, 56. The reduced activity

282

of FASN led to decreased levels of fatty acids in zebrafish following exposure to

283

propiconazole. Furthermore, the expression of genes associated with fatty acids β-

284

oxidation process were altered by propiconazole. The first step of acyl-CoA was

285

conversed to acyl-carnitine by cpt1 catalysis that transports fatty acids from the external

286

membrane into the mitochondrial, and the pparα induces these oxidations

287

pparα could be activated by free fatty acids or lipids, which reduces the rate of lipid

288

degeneration by increasing the rate of lipid metabolism in organisms 59, 60. In our study,

289

the reductions of fatty acids were in response to the down-regulation of cpt1 and pparα.

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The transcription factors screbf1 and chrebp are necessary regulators involved in fatty

291

acids synthesis. Glucose regulates the expression of chrebp gene, which stimulates the

292

process of lipogenesis 61. The apgat4 gene, codes the protein for catalyzing the glycerol

293

phosphate 62. DGAT, as the last step of TG biosynthesis, which catalyzes the conversion

294

of fatty acids and glycerol into triglyceride

295

catalyzes the decomposition of TG into fatty acids and monoglycerides in very low-

296

density lipoprotein (VLDL) to be used for tissue oxidation energy supply and storage

297

64.

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the up-regulation of screbf1, chrebp, apgat4, dgat2 expression and decreased LPL

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activity cause increased TG content in zebrafish larvae. Overall, we found that the

300

expression levels of genes related to fatty acid β-oxidation, lipogenesis, and cholesterol

301

metabolism were significantly increased and the enzyme activities of LPL and FASN

63.

57, 58.

The

In addition, LPL, as a glycoprotein,

The activity of LPL was decreased in zebrafish exposed to propiconazole. In general,

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was markedly decreased, suggesting that such changes resulted in reduced contents of

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fatty acids and disrupted lipid metabolism.

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Lipids are associated with biomembrane composition, energy metabolism, and other

305

physiological processes, which plays a crucial role in zebrafish development

306

According to the previous study, triazole fungicide difenoconazole could induce the

307

disruption of lipid metabolism and impair embryonic development on the basis of

308

transcriptomics and metabolomics by showing changes in expression levels of genes

309

involved in lipogenesis and lipolysis 17. Similarly, in this work, we observed that the

310

inhibition of developmental parameters were related to the disturbance of lipids

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metabolism.

312

Fatty acids are aliphatic acids and key components of cells membranes that play a key

313

role in energy transport and storage, cell structure, and intermediates that provide

314

hormone synthesis 66-69. Fatty acid metabolism usually takes place in mitochondria and

315

peroxisome. Saturated fatty acids may ameliorate environmental heat stress by

316

affecting mitochondrial energetics. Moreover, there is a close relationship between fatty

317

acids and cardiovascular function 70, 71. Locomotor activity, as a quantitative endpoint,

318

is used for measuring testing behavioral toxicity in aquatic organism72. The reduction

319

of fatty acids affected the energy metabolism and then further influenced locomotor

320

activity of embryo and heartbeat number, which may cause decreased heartbeat,

321

lowered average velocity, short in moving distance, more inactive time and less active

322

time on larvae after propiconazole exposure for 120 hpf, compared to the control group.

323

Previous studies have shown that fatty acids are involved in growth, cognition, and 16 ACS Paragon Plus Environment

65.

Page 17 of 40

Journal of Agricultural and Food Chemistry

324

stunting in the early development of organisms 73-75. In general, embryos have abundant

325

TG, TCHO, and phosphatidylcholine during embryogenesis and larval development

326

period from 0 hpf to 120 hpf

327

acids may inhibit the growth performance of zebrafish embryos following

328

propiconazole exposure, showing decreases in hatching rate, and body length.

329

Furthermore, the levels of lipid species are associated with fish quality and nutrient

330

retention 76. Therefore, these results indicate that we should pay great attention to the

331

impact of environmental pollutants on aquatic organisms.

332

In the present study, we demonstrate that exposure to propiconazole alters the

333

expression of genes associated with the lipogenesis and lipolysis pathway, such changes

334

leading to decreasing fatty acid synthesis, and enhancing cholesterol metabolism and

335

fatty acid β-oxidation, overall causing the disruption of lipid metabolism in zebrafish.

336

Both decreased activity of LPL and FASN and increased expression of lipolysis may

337

contribute to the reduction of TCHO and fatty acids in larvae. Taken together, our

338

results indicate that propiconazole is a contributing factor to the abnormal development

339

at the early life stages of organisms.

340

environmental and health risks of propiconazole should be considered, especially at the

341

early life stages of aquatic organisms.

342

Acknowledgements

343

Conflicts of interest

344

The authors declare that there are no conflicts of interest.

65.

However, in this report, decreased contents of fatty

Therefore, we suggest that the potential

17 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

345

Supporting Information

346

More experimental details, nucleotide sequences of primers, percentage recovery of

347

propiconazole, GC-MS instrumental parameters.

348

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Acknowledgements

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29 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure Captions Figure 1. Effects of propiconazole exposure on embryonic development in zebrafish. A. The number of spontaneous movements at 24 hpf. B. The number of heartbeat at 48 hpf. C. The hatching rate at 72, 96, and 120 hpf. D. The body length of hatched individual larvae at 120 hpf. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 2. Embryos with malformations following exposure to propiconazole at 120 hpf. A. Embryo in the control group; B/C. Embryo with spinal deformation (Sd) in the 2.5 mg/L group. D/F/G. Embryo with spinal deformation in the 4.5mg/L group. E. Embryo with tail malformation (Tm) in the 4.5mg/L propiconazole group. H. Malformation rate of 120 hpf larvae exposed to propiconazole. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 3. Locomotor activity of zebrafish larvae exposed to propiconazole at 120 hpf. A. The average velocity of zebrafish larvae at 120 hpf. B. The moving distance of zebrafish larvae at 120 hpf. C. Active and inactive time [s] of zebrafish larvae at 120 hpf. D. Average acceleration and deceleration of zebrafish larvae at 120 hpf. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation (SD)). Figure 4. Triglyceride (TG, A) and total cholesterol (TCHO, B) contents of zebrafish at 120 hpf following propiconazole exposure. * P < 0.05 compared with the control group, (n = 6 replicates, mean ±standard deviation). Figure 5. Lipoprtein lipase (LPL, A) and fatty acid synthase (FASN, B) activity of 30 ACS Paragon Plus Environment

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zebrafish at 120 hpf following propiconazole exposure. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 6. Propiconazole exposure induced the mRNA expression of genes involved in lipid metabolism in larvae at 120 hpf. A. The expression of genes involved in lipid accumulation. B. The expression of genes involved in lipogenesis. C. The expression of genes involved in fatty acid (FA) β-oxidation. D. The expression of genes involved in cholesterol metabolism. * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation). Figure 7. Effects of propiconazole exposure on the contents of free fatty acids in larvae at 120 hpf. A: saturated fatty acids (SFA); B: monounsaturated fatty acids (MUFA). * P < 0.05 compared with the control group, (n = 6 replicates, mean ± standard deviation).

31 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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Tables Table 1. The Report of Propiconazole Concentration in the Water Environment in Different Areas. Sampled sites

Concentration

References

Banana plantation sites Costa Rica

0.15–13 μg L−1

(Castillo et al. 2006)

Influent of pharmaceutical company

(Van De Steene and Lambert 0.17–0.24 μg L−1

Belgium

2008)

Effluent of pharmaceutical company

(Van De Steene and Lambert 0.012–0.14μg L−1

Belgium

2008)

Paris sewer France

0.15–0.21μg L−1

(Gasperi et al.2008)