Chemosphere 251 (2020) 126418

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Exposure to prothioconazole induces developmental toxicity and

cardiovascular effects on zebrafish embryo
Yongqi Sun a, Yi Cao a, Lili Tong a, Fangyi Tao a, Xiaonan Wang a, Huiming Wu a, *,
Mengcen Wang b, **
a
School of Agricultural and Food Science, Zhejiang Agriculture & Forestry University, Hangzhou, China
b
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Pesticide & Environmental Toxicology, Zhejiang
University, Hangzhou, China

h i g h l i g h t s

Prothioconazole was median toxic to zebrafish embryos.

Prothioconazole caused severe developmental abnormalities of zebrafish embryos.

Prothioconazole induced cardiovascular toxicity with deficient heart morphology and function.

The transcription of cardiac-related genes of embryos changed by prothioconazole.

a r t i c l e i n f o a b s t r a c t

Article history: Prothioconazole is a fungicide that has been widely used in general agriculture and livestock husbandry.
Received 20 December 2019 This study evaluated the acute toxicity of prothioconazole to zebrafish embryos by assessing their
Received in revised form hatching rate and malformation when exposed to different concentrations of prothioconazole. The 96 h-
29 February 2020
LC50 value of zebrafish embryos was 1.70 mg/L. Upon exposure to 0.85 mg/L, the mortality rate of the
Accepted 3 March 2020
Available online 7 March 2020
embryos significantly increased while their hatching rate decreased significantly. At prothioconazole
concentrations higher than 0.43 mg/L, developmental morphologic abnormalities such as heart and yolk-
Handling Editor: David Volz sac edema, spine curvature, tail deformity, shortened body length and decreased eye area were observed.
The heart rate of embryos decreased in a dose-dependent fashion during the exposure time. Prothio-
Keywords: conazole exposure also resulted in increased rates of cardiac malformation detected by significant in-
Prothioconazole crease in the distance between the sinus venosus and bulbus arteriosus and the pericardium area.
Developmental toxicity Moreover, the expression levels of genes related to cardiac development (amhc, vmhc, fli1, hand2, gata4,
Cardiovascular toxicity nkx2.5, tbx5 and atp2a2a) were significantly altered after exposure to prothioconazole. Indeed, this study
Zebrafish embryo
revealed the adverse effects on the developmental and cardiovascular system of zebrafish embryo caused
by prothioconazole. It further elucidated the risk of prothioconazole exposure to vertebrate cardiovas-
cular toxicity. As such, it provides a theoretical foundation for pesticide risk management measures.
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction growth has led to an increasing demand for agricultural products

Application of pesticides in crop fields protects and controls
them from diseases, pests and weeds thus guaranteeing the quality
of agricultural products (WHO, 2010). Worldwide population
* Corresponding author. School of Agricultural and Food Science, Zhejiang Agri-
culture & Forestry University, China.
** Corresponding author. Institute of Pesticide and Environmental Toxicology,
Zhejiang University, China.
E-mail addresses: wuhm@zafu.edu.cn (H. Wu), wmctz@zju.edu.cn (M. Wang).
thus necessitating the usage of pesticides (Xie et al., 2019b). Tri-
azole fungicides characterized by moderate lipophilicity and long-
term stability (Aladaghlo et al., 2019). As such, when they are used
without wholesome health supervision, they can enter the envi-
ronment and disperse in water bodies, causing toxic effects on non-
target aquatic life. (Kong et al., 2016; Satapute et al., 2019; Tong
et al., 2019). Previous studies reported the toxicity of triazole fun-
gicides, penconazole and imazalil inhibited the survival rate and
hatching rate of zebrafish embryos (Icoglu Aksakal and Ciltas, 2018;
Jin et al., 2016). Triazole fungicides could also lure developmental

https://doi.org/10.1016/j.chemosphere.2020.126418
0045-6535/© 2020 Elsevier Ltd. All rights reserved.
2 Y. Sun et al. / Chemosphere 251 (2020) 126418
abnormalities in zebrafish embryos. Propiconazole induced peri-
cardial edema and hypo-melanization in zebrafish (Souders et al.,
2019). Pericardial edema, yolk sac edema, tail deformation and
spine deformation were observed in cyproconazole-treated em-
bryos (Cao et al., 2019).
Cardiovascular disease (CVD) is the leading cause of death
worldwide (Collins et al., 2017). There is a close relationship be-
tween agrochemicals particle and cardiovascular diseases (Matome
et al., 2016). Exposure to penconazole (Cha^ abane et al., 2016) and
tebuconazole (Ben Othme ne et al., 2020) affects myocardial cells
normal functioning in male adult rats. Triadimefon can induce
cardiovascular toxicity and functional failure of the heart in
zebrafish (Liu et al., 2017a). Fenbuconazole exposure impacts the
transcription of genes, which are responsible for the cardiac
development and function in zebrafish larvae (Wu et al., 2018b).
Similarly, propiconazole blocks the cycle of blood cells in zebrafish
embryos (Souders et al., 2019).
Prothioconazole is triazole fungicide, that has been widely used
for foliar spray application to control plant diseases caused by
Fusarium and Pyricularia grisea pathogens in numerous crops and
vegetables (Wang et al., 2019b). A study to evaluate the risk
assessment of prothioconazole residues in Ukraine consumers
revealed that dietary intake of prothioconazole residues unlikely
concern to public health (Prodanchuk et al., 2016). A market
monitoring study of more than 150 samples about animal food in
China didn’t detect any prothioconazole residue in all samples (Liu
et al., 2017c). There is no published data of accurate value of
environmental concentrations about prothioconazole in water
body. However, pesticides residue on soil surface after field appli-
cation can be mobilized with surface runoff (Larsbo et al., 2016). In
high-rainfall areas, prothioconazole was used to preserve wheat
yield and keep economically beneficial (Sassenrath et al., 2019), the
potential effects caused by prothioconazole to aquatic life should be
a concern. Previous study detected the acute toxicity of prothio-
conazole to Daphnia magna, Chlorella pyrenoidosa, and Lemna mino,
and the EC50 were 2.68, 9.33 and 1.85 mg/L respectively (Zhai et al.,
2019). It is reported that exposure to prothioconazole disturbed
reproduction of zebrafish (Zhuo, 2018), it can also cause lipid per-
oxidation and induced oxidative damage in zebrafish (Tian et al.,
2019). Prothioconazole also caused altered expression of hepatic
cytochrome P450 in Chinese lizards (Eremias argus) (Xie et al.,
2019b). Theses evidence revealed that prothioconazole can result
in considerable impact, especially on aquatic organisms.
Zebrafish is well established as a model aquatic organism for
drug/pharmaceutical research and eco-toxicology studies to iden-
tify environmental pollutants (Alestro € m et al., 2006; He et al.,
2014). It has numerous suitable features that make it an ideal
vertebrate model. For instance, its high fecundity makes it easy to
carry out experiments. It also has transparent embryos that are easy
to contain thus, making microscopy works possible. In particular, its
transparency is favorable for evaluating development of the heart,
heartbeat and other physical motions (Bakkers, 2011; Liu and
Stainier, 2012).
In this study, wild-type zebrafish embryos were used to char-
acterize the potential eco-toxicological effects exerted by prothio-
conazole. Upon exposure to serial levels of prothioconazole, the
physiological, developmental and morphological alterations in
zebrafish were identified. Of great interest was the developmental
and cardiovascular toxicity caused by prothioconazole. An array of
representative genes involved in Ca2þ-ATPase, cardiac morpho-
genesis and conduction system development were detected using
real-time-quantitative PCR (q-PCR).
2. Materials and methods
2.1. Zebrafish husbandry and embryo collection
Wild-type AB strain zebrafish (Danio rerio) were obtained from
the China Zebrafish Resource Center (CZRC, Wuhan). Adult zebra-
fish (seven months) were maintained under a light/dark cycle of
14:10 h (Wu et al., 2018a), and maintained in a flow-through sys-
tem filled with dechlorinated and UV-sterilized tap water. The
temperature is maintained at 26.0e28.0  C, pH at 7.0e7.5, and
dissolved oxygen >6.5 mg/L. Embryos were collected from
spawning adults, by putting a male and two females in the same
spawning box overnight. Embryos were gathered within an hour of
spawning, rinsed with system water, and maintained at 27  C in the
incubator for further experiment.
2.2. Prothioconazole exposure
Prothioconazole stock solution was prepared by dissolving 96%
pure prothioconazole powder (CAS: 178,928-70-6, obtained from
Jiangxi Zhengbang Chemical Company Limited, China) in acetone
(Sinopharm Chemical Reagent Company Limited, China). The so-
lution was then stored in the dark at 4  C. Zebrafish treated with
0.01% acetone served as vehicle control. Working solutions were
prepared by dilution of stock solutions with system water, all
treatment groups contained the same concentration of acetone
(0.01%) and all test solutions were replaced every 24 h. To protect
the pesticide solutions from light-induced decomposition (Xing
et al., 2018), all experiments were performed at 27  C in the dark
(Liu et al., 2017b).
2.3. Determination of LC50
An acute toxicity test of prothioconazole was performed on
zebrafish embryos based on the Organization for Economic Co-
operation and Development (OECD) guideline (OECD, 1992).
Briefly, 2hour-post-fertilization (hpf) embryos were randomly
selected and transferred to twenty-four-well microplates (NEST,
Shanghai, China), each well contained an embryo and 2 mL solu-
tion. Based on pervious tests, prothioconazole doses of 0, 1, 1.5, 2,
2.5 and 3 mg/L were chosen for the acute toxicity test, 24 embryos
were exposed to 96 hpf in each of the 6 different concentrations of
prothioconazole. The number of dead embryos was then counted
after 24 h. Each experiment was repeated thrice, and the average
result picked. 50% lethal concentration (96 h-LC50) of the prothio-
conazole solution was then estimated using IBM SPSS Statistics 20.
2.4. Assessment of embryo development
According to our preliminary tests, 0.22, 0.43 and 0.85 mg/L of
prothioconazole (about 1/8 LC50, 1/4 LC50 and 1/2 LC50) were cho-
sen in the experiment. Loss of heartbeat and coagulation of the
embryos were used to determine the mortality of embryos in the
different concentrations of prothioconazole solution. The survival
and hatching rate of embryos at 24, 48 and 72 hpf, was then
examined (n ¼ 24). Embryonic development is usually complete at
72 hpf. As such morphological analysis of the embryos was done at
that time (n ¼ 24). Images of embryos were taken under a stereo-
microscope (Lecia, MC165 C, Germany) and screened for typical
phenotype, mainly including yolk-sac edema, spinal curvature, tail
deformity and cardiac defects. Malformation rate and hatching rate
calculated based on the formula below:
 Y. Sun et al. / Chemosphere 251 (2020) 126418
Malformation rate ¼ the number of malformed embryos=the number of survival embryos  100%;
Hatching rate ¼ the number of hatched embryos=the total number of embryos  100%:
Ten larvae from each group were anesthetized with 30 mg/L
tricaine (Aladdin, Shanghai, China), observed and photographed for
morphometric analysis under a stereomicroscope (Lecia, MC165 C,
Germany) fitted with a microscope camera (Lecia, MC190 HD,
Germany), and the body length, head area, eye area and pericardial
area of the larvae were then measured using Leica Application Suite
4.9 software. Each experiment was repeated three times.
2.5. Assessment of the effect of prothioconazole on the heart
At 24, 48, 72 and 96 hpf, 10 embryos were randomly selected
from each treatment group and their heart rates measured by
counting per 20 s under an inverted microscope (Nikon, Eclipse
TS100, Japan).
At 72 hpf, 10 embryos were randomly selected from each
treatment group and stained with an o-dianisidine solution to
measure the reduction of blood erythrocyte induced by prothio-
conazole., The stain solution was contained with 0.6 mg/mL o-
dianisidine (Aladdin, Shanghai, China), 0.01 M sodium acetate (pH
4.5), 0.65% H2O2 and 40% (v/v) ethanol. Briefly, embryos were
stained for 15min in the dark and washed three times by Dimethyl
sulfoxide (Liu et al., 2017a; Nasrallah et al., 2019). To quantify the
heart red blood cells (RBCs) intensity of zebrafish embryos, images
were taken under a Nikon Eclipse 80i microscope, and were pro-
cessed by Image-Pro Plus 6.0 software (Media Cybernetics,
Bethesda, MD) for calculating the average integrated optical density
(IOD) (S) for erythrocyte positive staining (Zhu et al., 2019). The
relative RBCs intensity ¼ [S(Pesticide treatment)/
S(Vehicle)]  100%.
Cardiac malformations were easily visualized under an inverted
microscope (Nikon, Eclipse TS100, Japan) assessed at 72 hpf. On the
other hand, the heart functioning and looping in zebrafish embryos
were investigated by measuring the distance between SV and BA.
The embryos were anesthetized by 30 mg/L tricaine and measured
by the NIS-Elements F3.10 image analysis software (Nikon, Japan)
(n ¼ 10). Each experiment was repeated three times.
2.6. Gene expression analysis
Total RNA was extracted from all the three biological replicates
of twenty embryos randomly selected from each treatment group
at 72 hpf by using UNlQ-10 Column Trizol Total RNA Isolation Kit
(Sangon, Shanghai, China). The manufacturer’s instructions were
followed without any modifications during extraction. The total
RNA was measured by a spectrophotometer (BioDrop, England) to
verify the quantity and purity, and the samples with a 260/280 nm
ratio between 1.9 and 2.0 were selected for further analysis.
The primer sequences of genes were shown in Table S2 (Sup-
plementary material). heavy chain 6 (amhc), ventricular myosin
heavy chain (vmhc), Fli-1 proto-oncogene (fli1), heart and neural
crest derivatives expressed 2 (hand2), GATA binding protein 4
(gata4), NK2 homeobox 5 (nkx2.5), T-box 5 (tbx5) and ATPase
sarcoplasmic/endoplasmic reticulum Ca2þ transporting 2a
(atp2a2a) were selected as cardiac development-related genes. The
3
amplification efficiencies of primers (Table S2) were determined by
constructing a standard curve of cycle threshold (Ct) values and
serially-diluted cDNA, and calculated by the equation: amplifica-
tion efficiencies ¼ 10(1/slope)-1. Melt curve analysis was performed
to demonstrate the specificity of the PCR product, as displayed by a
single peak in Figs. S1AeI b-actin was treated as a housekeeping
gene due to its stable expression among zebrafish embryos (Li et al.,
2018; Qian et al., 2020), and the results of RT-qPCR confirmed the
stability in the present study (Fig. S1J).
First-strand cDNA synthesis to convert the RNA to cDNA was
then done using M-MuLV First Strand cDNA Synthesis Kit (Sangon,
Shanghai, China). RT-qPCR amplification of representative genes
involved in cardiac development was done using the SYBR Mix
(Sangon, Shanghai, China) in 20 mL reaction volume on a real-time
PCR system (Biorad, CA, USA). All primers were synthesized by
Sangon (Shanghai, China). Thermal cycling conditions were set at
95  C for 30 s, followed by 39 cycles of 95  C for 10 s and 60C for
30 s. The expression levels of target genes were calculated by the
2DDCt method (Livak and Schmittgen, 2001). This was done in
three technical replicates.
2.7. Chemical analysis
Samples of freshly made prothioconazole solutions for each
treatment were collected thrice for the quantification of prothio-
conazole in water, which was confirmed by high-performance
liquid chromatography (HPLC, Shimadzu, Japan). This was also
repeated before the first replacement of the solutions. Filtrated
experimental solutions (0.22 mm) were injected directly into the
HPLC, and the analysis was done on a C18 column (4.6  250 mm,
5 mm) at 20  C and wavelength of 254 nm. The elution program was
performed with a mobile phase of acetonitrile and 0.1% glacial
acetic acid (70:30, v/v). The injection volume was 20 mL at a flow
rate of 2 mL/min. This was done in three technical replicates.
The concentration of prothioconazole was quantified by
measuring the area under the peak, which was drawn from the
analysis of eight known prothioconazole concentrations (from 0.1
to 10 mg/L) in Supporting Information (Fig. S2). The concentration
of prothioconazole in each sample was interpolated from the linear
correlation plot between the integrated areas and the concentra-
tion of the standard solutions.
2.8. Statistical analysis
All data were analyzed by the SPSS 20.0 software (SPSS, Chicago,
USA) and GraphPad Prism8 software (GraphPad Software Inc., USA).
One-way analysis of variances (ANOVA) was used followed by
Dunnett’s test to determine the statistical differences between the
treatment groups. A P value of less than 0.05 (P < 0.05) was
considered statistically significant.
4 Y. Sun et al. / Chemosphere 251 (2020) 126418
3. Results
3.1. Lethal effect of prothioconazole
The number of dead embryos was calculated at the end of
exposure time in each treatment group. The 96 h-LC50 value of
prothioconazole was 1.70 mg/1, with a 95% confidence interval of
between 1.33 and 1.82 mg/L (Table S1). Based on the toxicity
grading standards, prothioconazole is considered the median toxic
to zebrafish embryos (Cai, 2004; OECD, 1992).
3.2. Developmental toxicity to zebrafish embryo and larvae
Based on the chemical analysis, The deviation between the
measured concentration and the theoretical concentration was less
than 20% (Table S2), thus the nominal concentrations could
represent the actual concentrations in this study (OECD, 1992).
The mortality rate of treated zebrafish embryos in all concen-
trations and those in the control group at 24 and 48 hpf was not
significantly different (Fig. 1A). However, the mortality rate be-
tween the treatment groups and the control changed significantly
at 0.85 mg/L concentration of prothioconazole at 72 and 96 hpf.
The hatching rate at 48 hpf was 55.63 ± 12.37%, 32.85 ± 2.21%,
15.76 ± 13.34% and 1.521 ± 2.63% for the control, 0.22, 0.43 and
0.85 mg/L of prothioconazole respectively. At 72 hpf, the hatching
rate of the control group and at 0.22 mg/L of prothioconazole was
100%. However, there was a significant decrease in the hatching
rate at 0.85 mg/L of prothioconazole (Fig. 1B).
A change in the morphology of the zebrafish embryos was
detected at 72 hpf. Clear abnormalities such as yolk-sac edema,
spinal curvature, tail deformity and cardiac defects were observed
in prothioconazole-treated embryos (Fig. 2A). Moreover, the mal-
formation rate increased in a dose-dependent manner with a sig-
nificant hike in the malformation rate observed at 0.43 and
0.85 mg/L treatment groups in contrast to the control group. The
malformation rates of the different groups were: 9.72 ± 2.12%,
52.78 ± 3.21%, 76.39 ± 0.80% and 2.78 ± 0.80% for the control, 0.22,
0.43 and 0.85 mg/L treatment groups respectively (Fig. 2B). Ac-
cording to the accounts of malformation numbers in each group
Fig. 2C), pericardial edema was the most serious abnormality at
0.43 and 0.85 mg/L treatment groups.
The body length, head area, eye area, and pericardium area of
larvae in the different groups were measured at 72 hpf to deter-
mine the developmental toxicity of prothioconazole on larvae
(Fig. 3). The apparent morphological alterations were shortening of
Fig. 1. Mortality and hatching rate of zebrafish embryos induced after exposure to prothioconazole. (A) Mortality rate of embryos exposed to prothioconazole at 24, 48, 72 and 96
hpf. (B) Hatching rate of zebrafish embryos exposed to a series of prothioconazole doses at 48 and 72 hpf. Data are presented as mean ± SE (n ¼ 24 embryos in each concentration
replicated thrice). *P < 0.05, **P < 0.01, ***P < 0.001.
body length (Fig. 3A), reduction of eye area (Fig. 3C), and increase of
pericardium area (Fig. 3D). There was no significant change in head
area (Fig. 3B). At 72 hpf, shorter body length of larvae was observed
in the 0.43 and 0.85 mg/L treatment groups, and smaller eye area
can be seen in 0.85 mg/L group. Similarly, the pericardium area
increased with increasing prothioconazole concentration in the
treatment groups at 72 hpf, especially in 0.43 and 0.85 mg/L dose,
which was 0.0817 ± 0.0258 mm2 and 0.0920 ± 0.0291 mm2
respectively, while the area of the control group was
0.0257 ± 0.0025 mm2.
3.3. Cardiovascular toxicity phenotype
The heart rate of embryos was recorded at 24, 36, 48 and 72 hpf
to further assess the influence of prothioconazole on their cardiac
function. The heart rates for the control, 0.22, 0.43 and 0.85 mg/L
treatment groups at 72 hpf was: 73.67 ± 0.42, 69.93 ± 0.81,
64.87 ± 1.02 and 55.37 ± 0.60 beats peer 20 s respectively (Fig. 4A).
Prothioconazole exposure had significant effects on the heart rate
of embryos in each treatment group and time period. Similarly, the
intensity of red blood cells (RBCs) of the embryos was significantly
decreased at 72 hpf in the treatment groups compared to that of the
control group (Fig. 4BeC).
At 72 hpf, a series of alterations in the heart morphology were
observed in a dose-dependent fashion (Fig. 5A). These alterations
included visible pericardial edema and string-shaped heart
(Fig. 5B). In the control group, the hearts were S-shaped, and there
was no visible edema. In contrast, hearts of embryos exposed to
prothioconazole were string-like and elongated. Treatment with
prothioconazole induced a significant increase in the SV-BA dis-
tance (Fig. 5C, p < 0.001). The SV-BA distance in the control, 0.22,
0.43 and 0.85 mg/L treatment groups were: 136.90 ± 2.12 mm,
151.80 ± 3.74 mm, 222.07 ± 8.58 mm and 301.66 ± 8.10 mm
respectively.
3.4. Gene expression
Expression of genes related to cardiovascular development was
significantly altered after exposure to prothioconazole. The mRNA
levels of vmhc, fli1, gata4, nkx2.5 and atp2a2a were significantly
decreased in dose-dependent fashion compared to the control
group (Fig. 6). The expression of amhc was decreased 0.73 and 0.50
times in the 0.43 and 0.85 mg/L treatment groups respectively
compared to that of the control group. Similarly, the transcription
level of tbx5 was reduced in the 0.85 mg/L treatment group
 Y. Sun et al. / Chemosphere 251 (2020) 126418 5

Fig. 2. Malformation of zebrafish larvae exposed to different prothioconazole doses at 72 hpf. (A) Malformation rate caused by different dosage of prothioconazole in zebrafish
larvae at 72 hpf. (B) Representative images of the malformation in prothioconazole treated larvae (arrows exhibit abnormalities): yolk-sac edema (YSE), pericardial edema (PE),
spinal curvature (SC) and tail deformity (TD). (C) The rate of prevalence malformations zebrafish larvae exposed to prothioconazole at 72 hpf. Data presented as mean ± SE (n ¼ 24
embryos in each concentration replicated thrice). **P < 0.01, ***P < 0.001.

Fig. 3. Phenotypes of larvae after 72 h exposure of different concentrations. (A) Body length, (B) head area, (C) eye area, and (D) pericardium area. Boxes represent 25th and 75th
percentiles, and whiskers represent the Min to Max. Solid lines within the boxes indicated the medians. (n ¼ 10 embryos in each concentration replicated thrice). **P < 0.01;
***P < 0.001.

whereas that of hand2 was significantly up-regulated 1.16 times in
the 0.85 mg/L treatment group compared to that of the control
group.
4. Discussion
Prothioconazole has been widely used in diverse agricultural
systems since its official launch into the market in 2004. In China,
prothioconazole was first registered and commercialized in 2019.
Previous reports showed that prothioconazole had the potential
reproductive toxicity to vertebrate (Xie et al., 2019a; Zhuo, 2018).
Our study utilized zebrafish embryo-larval model to evaluate the
toxic effects of prothioconazole. The results indicated that exposure
to prothioconazole could induce developmental toxicity, lure
adverse cardiovascular effects and change the expression of vital
cardiac transcription factors.
prothioconazole is considered as the median toxic to zebrafish
embryos. Similarly, plenty triazole pesticides such as penconazole,
6 Y. Sun et al. / Chemosphere 251 (2020) 126418

Fig. 4. Effects of cardiac function of zebrafish embryos induced by prothioconazole. (A) Heart rate of zebrafish embryos at 24, 36, 48 and 72 hpf exposed to prothioconazole doses
(n ¼ 30). (B) Concentration dependent reduction of the heart red blood cells intensity (RBCs) showed in zebrafish embryos treated with prothioconazole. (C) The images of RBCs
intensity of zebrafish at 72 hpf. The red dashed boxes indicate zebrafish heart. Data are mean ± SE (n ¼ 10 in each concentration embryos replicated thrice). **P < 0.01; ***P < 0.001.
(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. Effect of prothioconazole on the heart morphology of zebrafish embryos. (A) Representative images of the alteration on the heart morphology induced by prothioconazole at
72 hpf. (V: ventricle; A: atrium) (B) Rate of cardiac malformation at 72 hpf. (C)Quantification of SV-BA distance as a measure of prothioconazole-induced morphology alteration of
zebrafish embryo heart at 72 hpf. Data are mean ± SE (n ¼ 10, three replicates each concentration). ***P < 0.001.

tebuconazole and difenoconazole are considered as median or low
toxic to zebrafish embryos (Icoglu Aksakal and Ciltas, 2018; Li et al.,
2020; Mu et al., 2015). In this study, the 96 h-LC50 of zebrafish
embryos was 1.70 mg/L. This result was consisted with that of
Zhuo’s study who reported an LC50 of 1.83 mg/L in prothioconazole
treated embryos (Zhuo, 2018).
Statistics indicated that both the survival and hatching rates of
the embryos were affected by prothioconazole. Moreover, yolk sac
edema and pericardial edema were two typical malformations of
zebrafish embryos induced by prothioconazole. In zebrafish,
hatching is a key stage in which embryos transition to larvae. The
epidermis of the yolk sac is where the hatching gland cells located
(Inohaya et al., 1997). The previous study reported that triazole
pesticides inhibit the hatching of zebrafish embryos by reducing
the secretion of hatching enzymes (De la Paz et al., 2017),
demonstrating the disordered gene expression involved in fatty
acid metabolism and the synthesis of cholesterol in zebrafish em-
bryos (Goetz and Dix, 2009; Hermsen et al., 2011; Morrison et al.,
2014). This could be the factor resulting in the yolk sac edema
and unsuccessful hatching caused by prothioconazole in zebrafish
embryos. Similarly, shorter larvae body lengths and smaller eyes
were detected as the prothioconazole doses increased. These re-
sults suggested that prothioconazole induced notochord and
craniofacial skeleton malformation thus adversely affecting the
development of zebrafish embryos and larvae (Felix et al., 2017; Wu
et al., 2018b). Moreover, some studies reported the available
mechanisms of which induce the malformation and disturb the
development of zebrafish embryos, including steroid biosynthesis
and retinol metabolism (Hermsen et al., 2012; Wu et al., 2018b). In
the same line, pericardial edema was the main types of
 Y. Sun et al. / Chemosphere 251 (2020) 126418
Fig. 6. Expression of cardiovascular developmental genes after exposed to prothioconazole at 72 hpf. (data are mean ± SE, determined by Dunnett post-hoc comparison, *p < 0.05,
**p < 0.01, ***p < 0.001).
malformations caused by prothioconazole, we determined the
impact of cardiovascular development on prothioconazole-treated
embryos.
Heart is the first organ to form. It plays a critical role during the
development of a zebrafish embryo (Fishman and Chien, 1997;
Stainier et al., 1993). The previous report indicated that the heart
rate was a key parameter in assessing cardiac function (Liu et al.,
2017a). They further reported that the heart rate of zebrafish em-
bryos was significantly decreased by prothioconazole during the
embryonic stage leading to heart failure occasion by triadimefon
poisoning. This study showed that prothioconazole could inhibit
the heart rate of zebrafish embryos. Heart contraction is initiated by
calcium (Ca2þ) influx into myocytes in zebrafish embryos. The
myocyte protein sarcoplasmic reticulum Ca2þ-ATPase2 (SERCA2a)
has to sequester calcium into the sarcoplasmic reticulum for the
continued rhythmical contractions to occur (Ebert et al., 2005). As
such, decreased expression of SERCA2a result in reduced Ca2þ
influx, leading to heart failure. Hence, the ATPase-related genes
(Atp2a2a) was down-regulated significantly in the treatment
groups at 72 hpf. These results were consistent with the previous
report (Wang et al., 2019a), when they did similar studies using 6-
benzyl amino purine as the treatment component.
Zebrafish larvae exposed to prothioconazole exhibited severe
cardiac malformation such as pericardial edema and elongation of
the heart. These toxicity effects of prothioconazole were consistent
with those of other compounds reported in other studies such as
acrylamide (Huang et al., 2018), DBP (Sun and Li, 2019) and mac-
rolides (Yan et al., 2019). The SV-BA distance in the heart also fell
outside the normal range accordingly. This occurred in the previous
report when treated by triadimefon in zebrafish embryos, in which
they speculated severe pericardial edema led to the mechanical
stretching of the hearts indirectly (Liu et al., 2017a). Correct looping
is essential for the formation of a normal heart structure
(Antkiewicz et al., 2005; Ramasubramanian et al., 2008). Zebrafish
embryo/larvae that were exposed to prothioconazole had mis-
positioned atria and ventricles during heart development. These
results implied that prothioconazole targets the heart of zebrafish
by causing a detrimental impact on its development.
Gene expression profiles indicated that several key genes
related to cardiac development were altered by prothioconazole
toxicity. The expression of myosin heavy chains genes amhc and
vmhc and that of vascular endothelial cell gene fli1 was significantly
down-regulated in prothioconazole-exposed embryos. This was an
indication that prothioconazole could disrupt heart chamber for-
mation and inhibit angiogenesis (Li et al., 2019; Shih et al., 2015;
7
Stainier, 2001). On the other hand, hand2 was up-regulated,
possibly as a stress response in an attempt to regenerate the pro-
liferation of cardiomyocytes ameliorate the injury caused by pro-
thioconazole (Huang et al., 2019; Schindler et al., 2014).
Homeodomain transcription factor nkx2.5 and zinc-finger
transcription factor gata4 are essential for heart formation. They
are early markers of precardiac cells (Durocher et al., 1997). On the
other hand, the T-box transcription factor tbx5 plays a pivotal role
in development and maturation of the cardiac conduction system
(Moskowitz et al., 2004). Mutations in nkx2.5 and tbx5 have been
reported to occur leading to human Coronary Heart Disease (CHD)
and malformed cardiac conduction system in mice (Ellesøe et al.,
2016; Jay et al., 2004; Moskowitz et al., 2004; Pashmforoush
et al., 2004). Similarly, the loss of gata4 can cause the interrup-
tion of late cardiac morphogenetic movements (Holtzinger and
Evans, 2005; Liu et al., 2017b). Down-regulation of these genes by
prothioconazole affected the cardiogenic differentiation program,
consequently leading to cardiac malformation in zebrafish
embryos.
5. Conclusion
Evidently, this study revealed numerous functional and struc-
tural impairments caused by prothioconazole during embryo and
larvae development of zebrafish. It further elucidated the risk of
prothioconazole exposure to vertebrate cardiovascular toxicity. As
such, it provides a theoretical foundation for pesticides risk man-
agement measures.
Authors’ contributions
Mengcen Wang and Huiming Wu conceived and designed the
study. Yongqi Sun, Yi Cao, Lili Tong, Fangyi Tao and Xiaonan Wang
performed the experiments. Huiming Wu provided the funding and
Yongqi Sun wrote the manuscript. All authors read and approved
the manuscript. Authors claimed the work has not been published
previously.
Declaration of competing interest
The authors have declared no conflict of interests.
Acknowledgments
This work was supported by the Natural Science Foundation of
8 Y. Sun et al. / Chemosphere 251 (2020) 126418
Zhejiang Province (no. LY15B070008) and “Sannong Liufang” Sci-
ence & Technique Cooperation Program of Agriculture and Rural
Department of Zhejiang Province (no. CTZB-F180706LWZ-SNY1).
The authors would like to thank Dr. Qian Shen (Thomas Jefferson
University Hospital, Philadelphia) for her generous guides in aca-
demic writing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.chemosphere.2020.126418.
References
Aladaghlo, Z., Fakhari, A.R., Alavioon, S.I., Dabiri, M., 2019. Ultrasound assisted
dispersive solid phase extraction of triazole fungicides by using an N-hetero-
cyclic carbene copper complex supported on ionic liquid-modified graphene
oxide as a sorbent. Microchimica Acta 186, 209.
Alestro€ m, P., Holter, J.L., Nourizadeh-Lillabadi, R., 2006. Zebrafish in functional ge-
nomics and aquatic biomedicine. Trends Biotechnol. 24, 15e21.
Antkiewicz, D.S., Burns, C.G., Carney, S.A., Peterson, R.E., Heideman, W., 2005. Heart
malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci.
84, 368e377.
Bakkers, J., 2011. Zebrafish as a model to study cardiac development and human
cardiac disease. Cardiovasc. Res. 91, 279e288.
Ben Othme ne, Y., Hamdi, H., Annabi, E., Amara, I., Ben Salem, I., Neffati, F., et al.,
2020. Tebuconazole induced cardiotoxicity in male adult rat. Food Chem. Tox-
icol. 137, 111134.
Cai, D., 2004. Testing Guidelines of Assessing Environmental Safety of Chemical
Pesticides. State Environmentla Protection Administration of China, Beijing.
Cao, F., Souders, C.L., Li, P., Pang, S., Qiu, L., Martyniuk, C.J.J.E.S., et al., 2019. Devel-
opmental toxicity of the triazole fungicide cyproconazole in embryo-larval
stages of zebrafish (Danio rerio). Environ. Sci. Pollut. Control Ser. 26,
4913e4923.
Cha^abane, M., Tir, M., Hamdi, S., Boudawara, O., Jamoussi, K., Boudawara, T., et al.,
2016. Improvement of heart redox states contributes to the beneficial effects of
selenium against penconazole-induced cardiotoxicity in adult rats. Biol. Trace
Elem. Res. 169, 261e270.
Collins, D.R.J., Tompson, A.C., Onakpoya, I.J., Roberts, N., Ward, A.M., Heneghan, C.J.,
2017. Global cardiovascular risk assessment in the primary prevention of car-
diovascular disease in adults: systematic review of systematic reviews. BMJ
open 7 e013650.
De la Paz, J.F., Beiza, N., Paredes-Zún ~ iga, S., Hoare, M.S., Allende, M.L., 2017. Triazole
fungicides inhibit zebrafish hatching by blocking the secretory function of
hatching gland cells. Int. J. Mol. Sci. 18, 710.
Durocher, D., Charron, F., Warren, R., Schwartz, R.J., Nemer, M., 1997. The cardiac
transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J. 16,
5687e5696.
Ebert, A.M., Hume, G.L., Warren, K.S., Cook, N.P., Burns, C.G., Mohideen, M.A., et al.,
2005. Calcium extrusion is critical for cardiac morphogenesis and rhythm in
embryonic zebrafish hearts. Proc. Natl. Acad. Sci. U.S.A. 102, 17705e17710.
Ellesøe, S.G., Johansen, M.M., Bjerre, J.V., Hjortdal, V.E., Brunak, S., Larsen, L.A., 2016.
Familial atrial septal defect and sudden cardiac death: identification of a novel
NKX2-5 mutation and a review of the literature. Congenit. Heart Dis. 11,
283e290.
lix, L.M., Serafim, C., Martins, M.J., Valentim, A.M., Antunes, L.M., Matos, M., et al.,
Fe
2017. Morphological and behavioral responses of zebrafish after 24h of keta-
mine embryonic exposure. Toxicol. Appl. Pharmacol. 321, 27e36.
Fishman, M.C., Chien, K.R., 1997. Fashioning the vertebrate heart: earliest embryonic
decisions. Development 124, 2099e2117.
Goetz, A.K., Dix, D.J., 2009. Toxicogenomic effects common to triazole antifungals
and conserved between rats and humans. Toxicol. Appl. Pharmacol. 238, 80e89.
He, J.-H., Gao, J.-M., Huang, C.-J., Li, C.-Q., 2014. Zebrafish models for assessing
developmental and reproductive toxicity. Neurotoxicol. Teratol. 42, 35e42.
Hermsen, S.A.B., Pronk, T.E., van den Brandhof, E.-J., van der Ven, L.T.M.,
Piersma, A.H., 2011. Chemical class-specific gene expression changes in the
zebrafish embryo after exposure to glycol ether alkoxy acids and 1,2,4-triazole
antifungals. Reprod. Toxicol. 32, 245e252.
Hermsen, S.A.B., Pronk, T.E., van den Brandhof, E.-J., van der Ven, L.T.M.,
Piersma, A.H., 2012. Triazole-induced gene expression changes in the zebrafish
embryo. Reprod. Toxicol. 34, 216e224.
Holtzinger, A., Evans, T., 2005. Gata4 regulates the formation of multiple organs.
Development 132, 4005e4014.
Huang, M., Jiao, J., Wang, J., Xia, Z., Zhang, Y., 2018. Characterization of acrylamide-
induced oxidative stress and cardiovascular toxicity in zebrafish embryos.
J. Hazard Mater. 347, 451e460.
Huang, M., Zhu, F., Jiao, J., Wang, J., Zhang, Y., 2019. Exposure to acrylamide disrupts
cardiomyocyte interactions during ventricular morphogenesis in zebrafish
embryos. Sci. Total Environ. 656, 1337e1345.
Icoglu Aksakal, F., Ciltas, A., 2018. Developmental toxicity of penconazole in Zebrfish
(Danio rerio) embryos. Chemosphere 200, 8e15.
Inohaya, K., Yasumasu, S., Araki, K., Naruse, K., Yamazaki, K., Yasumasu, I., et al., 1997.
Species-dependent migration of fish hatching gland cells that commonly ex-
press astacin-like proteases in common. Dev. Growth Differ. 39, 191e197.
Jay, P.Y., Harris, B.S., Maguire, C.T., Buerger, A., Wakimoto, H., Tanaka, M., et al., 2004.
Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system.
J. Clin. Invest. 113, 1130e1137.
Jin, Y., Zhu, Z., Wang, Y., Yang, E., Feng, X., Fu, Z., 2016. The fungicide imazalil induces
developmental abnormalities and alters locomotor activity during early
developmental stages in zebrafish. Chemosphere 153, 455e461.
Kong, Z., Li, M., Chen, J., Bao, Y., Fan, B., Francis, F., et al., 2016. Processing factors of
triadimefon and triadimenol in barley brewing based on response surface
methodology. Food Contr. 64, 81e86.
Larsbo, M., Sandin, M., Jarvis, N., Etana, A., Kreuger, J., 2016. Surface runoff of pes-
ticides from a clay loam field in Sweden. J. Environ. Qual. 45, 1367e1374.
Li, H., Cao, F., Zhao, F., Yang, Y., Teng, M., Wang, C., et al., 2018. Developmental
toxicity, oxidative stress and immunotoxicity induced by three strobilurins
(pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos. Che-
mosphere 207, 781e790.
Li, M., Liu, X., Feng, X., 2019. Cardiovascular toxicity and anxiety-like behavior
induced by deltamethrin in zebrafish (Danio rerio) larvae. Chemosphere 219,
155e164.
Li, S., Jiang, Y., Sun, Q., Coffin, S., Chen, L., Qiao, K., et al., 2020. Tebuconazole induced
oxidative stress related hepatotoxicity in adult and larval zebrafish (Danio
rerio). Chemosphere 241, 125129.
Liu, H-c, Chu, T-y, Chen, L-l, Gui, W-j, Zhu, G-n, 2017a. The cardiovascular toxicity of
triadimefon in early life stage of zebrafish and potential implications to human
health. Environ. Pollut. 231, 1093e1103.
Liu, H., Chu, T., Chen, L., Gui, W., Zhu, G., 2017b. In vivo cardiovascular toxicity
induced by acetochlor in zebrafish larvae. Chemosphere 181, 600e608.
Liu, H., Yao, G., Liu, X., Liu, C., Zhan, J., Liu, D., et al., 2017c. Approach for pesticide
residue analysis for metabolite prothioconazole-desthio in animal origin food.
J. Agric. Food Chem. 65, 2481e2487.
Liu, J., Stainier, D.Y.R., 2012. Zebrafish in the study of early cardiac development.
Circ. Res. 110, 870e874.
Livak, Kenneth J, Schmittgen, Thomas D, 2001. Analysis of relative gene expression
data using real-time quantitative PCR and the 2 DDCT method. Methods 25
(4), 402e408. https://doi.org/10.1006/meth.2001.1262.
Matome, S., Kotsedi, M., Masezi, S., 2016. Exposure to agrochemicals and cardio-
vascular disease: a review. Int. J. Environ. Res. Publ. Health 13, 229.
Morrison, A.M.S., Goldstone, J.V., Lamb, D.C., Kubota, A., Lemaire, B., Stegeman, J.J.,
2014. Identification, modeling and ligand affinity of early deuterostome CYP51s,
and functional characterization of recombinant zebrafish sterol 14a-demethy-
lase. Biochim. Biophys. Acta Gen. Subj. 1840, 1825e1836.
Moskowitz, I.P.G., Pizard, A., Patel, V.V., Bruneau, B.G., Kim, J.B., Kupershmidt, S.,
et al., 2004. The T-Box transcription factor Tbx5 is required for the patterning
and maturation of the murine cardiac conduction system. Development 131,
4107e4116.
Mu, X., Chai, T., Wang, K., Zhang, J., Zhu, L., Li, X., et al., 2015. Occurrence and origin
of sensitivity toward difenoconazole in zebrafish (Danio reio) during different
life stages. Aquat. Toxicol. 160, 57e68.
Nasrallah, G.K., Salem, R., Da’as, S., Al-Jamal, O.L.A., Scott, M., Mustafa, I., 2019.
Biocompatibility and toxicity of novel iron chelator Starch-Deferoxamine (S-
DFO) compared to zinc oxide nanoparticles to zebrafish embryo: an oxidative
stress based apoptosis, physicochemical and neurological study profile. Neu-
rotoxicol. Teratol. 72, 29e38.
OECD, 1992. OECD Guideline for Testing of Chemicals. Test No. 203: Acute Fish Test.
Organization for Economic Cooperation and Development Paris, France.
Pashmforoush, M., Lu, J.T., Chen, H., Amand, T.S., Kondo, R., Pradervand, S., et al.,
2004. Nkx2-5 pathways and congenital heart disease: loss of ventricular
myocyte lineage specification leads to progressive cardiomyopathy and com-
plete heart block. Cell 117, 373e386.
Prodanchuk, M., Kravchuk, O., Leposhkin, I., Nedopytanska, N., Zhminko, P.,
Ivanova, L., et al., 2016. Risk assessment of prothioconazole residues for
Ukrainian consumer. Toxicol. Lett. 258, S227eS228.
Qian, L., Liu, J., Lin, Z., Chen, X., Yuan, L., Shen, G., et al., 2020. Evaluation of the spinal
effects of phthalates in a zebrafish embryo assay. Chemosphere 126144.
Ramasubramanian, A., Nerurkar, N.L., Achtien, K.H., Filas, B.A., Voronov, D.A.,
Taber, L.A., 2008. On modeling morphogenesis of the looping heart following
mechanical perturbations. J. Biomech. Eng. 130.
Sassenrath, G.F., Farney, J., Lollato, R., 2019. Impact of fungicide and insecticide use
on wheat production in a high-rainfall environment. Crop, Forage & Turfgrass
Management 5, 190008.
Satapute, P., Kamble, M.V., Adhikari, S.S., Jogaiah, S., 2019. Influence of triazole
pesticides on tillage soil microbial populations and metabolic changes. Sci. Total
Environ. 651, 2334e2344.
Schindler, Y.L., Garske, K.M., Wang, J., Firulli, B.A., Firulli, A.B., Poss, K.D., et al., 2014.
Hand2 elevates cardiomyocyte production during zebrafish heart development
and regeneration. Development 141, 3112e3122.
Shih, Y.H., Zhang, Y., Ding, Y., Ross, C.A., Li, H., Olson, T.M., et al., 2015. Cardiac
transcriptome and dilated cardiomyopathy genes in zebrafish. Circulation:
Cardiovascular Genetics 8, 261e269.
Souders, C.L., Xavier, P., Perez-Rodriguez, V., Ector, N., Zhang, J.-L., Martyniuk, C.J.,
2019. Sub-lethal effects of the triazole fungicide propiconazole on zebrafish
(Danio rerio) development, oxidative respiration, and larval locomotor activity.
 Y. Sun et al. / Chemosphere 251 (2020) 126418
Neurotoxicol. Teratol. 74, 106809.
Stainier, D.Y.R., 2001. Zebrafish genetics and vertebrate heart formation. Nat. Rev.
Genet. 2, 39e48.
Stainier, D.Y.R., Lee, R.K., Fishman, M.C., 1993. Cardiovascular development in the
zebrafish: I. Myocardial fate map and heart tube formation. Development 119,
31e40.
Sun, G., Li, Y., 2019. Exposure to DBP induces the toxicity in early development and
adverse effects on cardiac development in zebrafish (Danio rerio). Chemo-
sphere 218, 76e82.
Tian, S., Teng, M., Meng, Z., Yan, S., Jia, M., Li, R., et al., 2019. Toxicity effects in
zebrafish embryos (Danio rerio) induced by prothioconazole. Environ. Pollut.
255, 113269.
Tong, Z., Dong, X., Yang, S., Sun, M., Gao, T., Duan, J., et al., 2019. Enantioselective
effects of the chiral fungicide tetraconazole in wheat: fungicidal activity and
degradation behavior. Environ. Pollut. 247, 1e8.
Wang, W., Wang, B., Liu, Z., Xia, X., 2019a. Developmental toxicity and alteration of
gene expression in zebrafish embryo exposed to 6-benzylaminopurine. Che-
mosphere 233, 336e346.
Wang, X., Liu, Y., Xue, M., Wang, Z., Yu, J., Guo, X., 2019b. Enantioselective degra-
dation of chiral fungicides triticonazole and prothioconazole in soils and their
enantioselective accumulation in earthworms Eisenia fetida. Ecotoxicol. Envi-
ron. Saf. 183, 109e491.
WHO, 2010. International Code of Conduct on the Distribution and Use of Pesti-
cides: Guidelines for the Registration of Pesticides. World Health Organization.
Wu, H., Rao, Q., Zheng, J., Mao, C., Sun, Y., Gu, D., et al., 2018a. Biochemical and
histological alterations in adult zebrafish (Danio rerio) ovary following expo-
sure to the tetronic acid insecticide spirotetramat. Ecotoxicol. Environ. Saf. 164,
9
149e154.
Wu, Y., Yang, Q., Chen, M., Zhang, Y., Zuo, Z., Wang, C., 2018b. Fenbuconazole
exposure impacts the development of zebrafish embryos. Ecotoxicol. Environ.
Saf. 158, 293e299.
Xie, Y., Jiang, H., Chang, J., Wang, Y., Li, J., Wang, H., 2019a. Gonadal disruption after
single dose exposure of prothioconazole and prothioconazole-desthio in male
lizards (Eremias argus). Environ. Pollut. 255, 113e297.
Xie, Y., Li, L.Y.Z., Hao, W., Chang, J., Xu, P., Guo, B., et al., 2019b. Comparative tox-
icokinetics and tissue distribution of prothioconazole and prothioconazole-
desthio in Chinese lizards (Eremias argus) and transcriptional responses of
metabolic-related genes. Environ. Pollut. 247, 524e533.
Xing, S., XiaoLong, Y., BangBao, Y., Yue, D., XiangYang, Y.J.S.K.F.S., 2018. Degradation
Dynamics and Residue Analysis of Prothioconazole and its Metabolite
Prothioconazole-Desthio in Wheat and Soil by QuEChERS-High Performance
Liquid Chromatography-Tandem Mass Spectrometry, vol. 39, pp. 269e275.
Yan, Z., Huang, X., Xie, Y., Song, M., Zhu, K., Ding, S., 2019. Macrolides induce severe
cardiotoxicity and developmental toxicity in zebrafish embryos. Sci. Total En-
viron. 649, 1414e1421.
Zhai, W., Zhang, L., Cui, J., Wei, Y., Wang, P., Liu, D., et al., 2019. The biological ac-
tivities of prothioconazole enantiomers and their toxicity assessment on
aquatic organisms. Chirality 31, 468e475.
Zhu, X.-Y., Xia, B., Wu, Y.-Y., Yang, H., Li, C.-Q., Li, P., 2019. Fenobucarb induces heart
failure and cerebral hemorrhage in zebrafish. Aquat. Toxicol. 209, 34e41.
Zhuo, L., 2018. Effect of Prothioconazole on the Growth and Reproduction of
Zebrafish. Hunan Agricultural University. http://cdmd.cnki.com.cn/Article/
CDMD-10537-1019876748.htm.