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Disruption of zebrafish (Danio rerio) sexual development after full life-cycle exposure to environmental levels of triadimefon Shao-ying Liu a , Quan Jin a , Xi-hui Huang a , Guo-nian Zhu b,∗ a
Laboratory of Chemistry and Physics, Hangzhou Center for Disease Control and Prevention, Hangzhou 310021, PR China b Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
In this study, zebrafish was exposed to environmental levels of triadimefon (0.125, 0.25,
Received 27 August 2013
0.5 g/mL) from 24 h post fertilization to 120 days post fertilization. Several endpoints that
Received in revised form
related to reproductive function were evaluated. It was found that the body length, body
31 October 2013
weight and vitellogenin transcription were significantly reduced for fish exposed to tri-
Accepted 7 November 2013
adimefon. Histological examination showed that the sex ratio of fish skewed to male and
Available online 1 December 2013
female exposed to 0.5 g/mL triadimefon had immature ovary. The breeding success, as determined from data on egg production and spawning, was reduced in fish exposed to
Keywords:
0.25 g/mL triadimefon. In the offspring, the reduced egg fertility, hatching rate and sur-
Triadimefon
vival were observed in eggs exposed to 0.5 g/mL triadimefon. These findings indicated
Zebrafish
that triadimefon had the potential to adversely affect the sexual development and breeding
Sexual development
success through the multiple endocrine actions.
Breeding success
© 2014 Elsevier B.V. All rights reserved.
Endocrine disrupting compounds
1.
Introduction
There are increasing evidences that a number of environmental substances have the potential to disrupt the endocrine systems of exposed organisms. Many of these socalled endocrine disrupting compounds (EDCs) interfere with the sexual development and reproduction (Rasmussen and Korsgaard, 2004). The potential risk posed by EDCs for human and wildlife has instigated efforts to establish regulatory programmes for EDC hazard assessment. Since the aquatic environment is a major sink for EDCs, a particular attention is given to the development and validation of testing approaches using fish. Zebrafish (Danio rerio) is under consideration as
a test species. Zebrafish develops from the fertilized egg to mature adults within a few months and is therefore perform full life-cycle tests with a variety of developmental and reproductive endpoints that might be affected by EDCs (Ankley and Johnson, 2004). Chemicals may adversely influence endocrine function via several pathways. For example, substances that affect the biosynthesis and metabolism of sex steroids can disturb function of the hypothalamic–pituitary–gonadal axis. In fish, the hormonal balance between estrogens and androgens appears to be an important factor in the course of sexual differentiation. For example, exposure of zebrafish to estrogens during early development has been shown to change the sex ratio towards more females or more males, respectively
∗ Corresponding author at: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Kaixuan Road 172, Hangzhou, Zhejiang 310029, PR China. Fax: +86 571 8643 0193. E-mail address: [email protected] (G.-n. Zhu). 1382-6689/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.11.007
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(Scholz and Kluver, 2009; Sofikitis et al., 2008). The balance of the sex hormone depends on the availability and activity of the steroid synthesizing enzymes, and particularly on the CYP19 aromatase, which catalyses the conversion of androgens to estrogens. The antifungal action of triadimefon is based on the inhibition of the cytochrome P450-dependent 14␣-demethylase required in the biosynthesis of ergosterol, a fungal cell membrane (Georgopapadakou and Walsh, 1996). However, triadimefon also modulates the activity of other cytochrome P450-dependent enzymes, including enzymes involved in vertebrate biosynthesis of steroids as for example CYP19 aromatase (Zarn et al., 2003). Triadimefon has a potential endocrine disruption effect from reports of European Union (EU) and World Wide Fund for Nature (WWF) in 2006 (UK, 2006). Environmental triadimefon concentrations in water are highly variable, from 0.046 to 0.922 g/mL (Watschke et al., 2000). From other study, the majority of the measured concentrations are below 0.3 g/mL (Vincelli, 2004). Triadimefon has the ability to bioconcentration in fish tissue (EPA, 2006). In the model teleost, zebrafish (Danio rerio), none paper has been conducted to illustrate the impact on reproductive parameters cover the full life-cycle to triadimefon. Therefore, the main aim of the present study was to examine the concentration-dependent effects of zebrafish full life-cycle exposure to low levels of triadimefon on the sexual development. In order to achieve this, zebrafish were exposed to low levels of triadimefon (0.125, 0.25, 0.5 g/mL), from 24 h post fertilization (hpf) to 120 days post fertilization (dpf). During this chronic exposure, some parameters of sexual development, such as gonad histological examination and sex ratios were investigated.
2.
Materials and methods
2.1.
Chemicals
Triadimefon (96%) was purchased from Bayer Crop Science. The stock solution of 5 mg/mL triadimefon was prepared in acetone and stored in darkness. Test solutions were prepared by dilution of the stock solution with the test water (aerated tap water), hardness (86 mg/L), pH value (6.5–7.5). The final concentration of acetone was not exceeding 0.01%, which was inactive to zebrafish (Hallare et al., 2006).
2.2.
Experimental animals and embryos collections
Adult wild-type zebrafish were maintained at 28 ± 1 ◦ C under a light/dark cycle of 14 h:10 h. Embryos were obtained from healthy adult fish with the sex ratio of 1:1. Spawning was induced in the next morning when the light was turned on. Fertilized and normal embryos were collected within 0.5 h of spawning and rinsed in the test water twice.
2.3.
Full life-cycle exposure of parental (F0) generation
About 50 embryos of 24 h post fertilization (hpf) were exposed to triadimefon (0.125, 0.25, 0.5 g/mL) and solvent control (0.01%) in 1 L water tanks. Triplicate exposures were performed
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for each test chemical concentration and solvent control. Every 48 h from 24 hpf to 120 dpf, a 100% water change was performed and fry were transferred to clean, acetone rinsed tanks. Fresh acetone or test chemicals were added at the time of the water change. Mortality and any abnormalities in appearance of surviving fish were observed and recorded daily over the 120-day exposure period. Dead fish were removed and discarded according to animal care protocols. At 40 dpf, 5 fish from each treatment and control were randomly selected for Vtg determination and there were three replications for each exposure concentration. Starting at exposure day 90 and concluding on exposure day 120, breeding trials were carried out to estimate the breeding success. At study termination, the fish from all replicates of a given treatment were pooled. The fish from each pooled group were selected for measurement of lengths, weights, sex ratio, sperm examination and gonad histology.
2.4. Physiological and biochemical indexes of parental (F0) generation 2.4.1.
Histological examination
At 120 dpf, the fish from all replicates of a given treatment group were pooled. The fish from each pooled group were randomly selected for measurement of weights and lengths. The gonads were get out and the gonadal somatic index (GSI) (gonad weight(g)/body weight(g) × 100) and the sex ratios were calculated. Gonads were fixed with 2.5% glutaraldehyde in phosphate buffer and then embedded in paraffin and 2 m sectioned were cut longitudinally through the gonad. The sections were stained with 0.1% methylthionine chloride and examined by light microscopy.
2.4.2.
Plasma sex hormones
At 120 dpf, blood samples from individual were collected from the caudal vein. There were three replicates for the control and the exposure treatments. 17-estradiol (E2) and testosterone (T) were detected using hormone detection kits (RD Company, USA), following the manufacturer’s instructions. The assay detection limit for T was 0.216 ng/mL and 0.100 ng/mL for E2.
2.4.3.
Sperm quality parameters in F0 males
At 120 dpf, the sperm from the male fish in each treatment were collected and diluted with HBSS 300 to achieve sperm suspension. Sperm concentration and motility were determined via a computer-assisted sperm analysis system (CASA, IVOS version 12.0, Hamilton Thorne Bioscience, Beverly, MA, USA). Five to six fields were analyzed and each of the sperm samples was tested in triplicate.
2.4.4.
Vtg mRNA expression
In this study, Vtg mRNA expression of zebrafish (38 dpf) was determined using quantitative real-time PCR. Total RNA was extracted from 5 homogenized fish by using Trizol Reagent (Invitrogen, Carlsbad CA, USA) according to the manufacturer’s instructions. To remove the genomic DNA contamination, the total RNA mixture was treated with RNaefree DNase I (Promega Madison, WI, USA) and then purified. Approximately 2 g RNA was used for cDNA Synthesis with M-MLV RTase (Takara Biochemicals, Dalian, China) according
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2.5.
F0 breeding trials and quality of offspring F1
During breeding trials conducted between 90 and 120 dpf, six randomly selected fish from each exposure concentration were placed into a breeding chamber in triadimefon (0.125, 0.25, 0.5 g/mL) and solvent control (0.01%) continuously. Three replicates of each exposure concentration were used for each trial. Fish were bred for 20 consecutive days with eggs being collected each day. The total number of eggs and spawning per female were used to assess the reproductive ability of parental (F0) generation. Then F1 embryos were maintained in the clean water until 120 hpf, the embryo fertility, mortality, hatching rate and malformation were monitored to assess the quality of offspring.
2.6.
Statistical analysis
One-way ANOVA was applied to calculate statistical significance followed by Dunnett’s test as a post hoc test to independently compare each exposure group to the control group. The LSD test was used as a post hoc test for multiple comparisons between groups. *P < 0.05 and **P < 0.01 were regarded as statistically significant using SPSS 16.0 (SPSS, Chicago, IL, USA). Data were presented as means ± standard error (Semenza et al., 1997).
3.
Results
3.1.
Survival and growth at 120 dpf
The cumulative mortalities, from 24 hpf to 120 dpf, varied between 64% and 80% and no differences were observed between control and triadimefon exposure groups (data not shown). These mortality rates were within the normal expected values for zebrafish (Soares et al., 2009). The weight and length of female fish exposed to 0.5 g/mL triadimefon were significantly lower than those of the control at 120 dpf (Table 1). Meanwhile, male fish exposed to
Male
Female *
100
80
60
%
to manufacturer’s description. The product from the reverse transcription was diluted 25 times and 5 L of each diluted sample, corresponding to 10 ng of the reverse transcribed RNA, was used for the real-time PCR. Quantitative real-time polymerase chain reaction (PCR) was performed by using the SYBR Green PCR kit (Takara Biochemicals, Dalian, China). Primers for Vtg1 and housekeeping gene (-actin) were performed as described previously by Martyniuk et al. (2007): Vtg1 upstream primer 5 -TCCATTGCTGAAAACGACAA-3 ; downstream primer 5 -GGCAGTCTCTCCCATTCCAC-3 ; actin upstream primer 5 -CGAGCAGGAGATGGGAACC-3 ; and -actin downstream primer 5 -CAACGGAAACGCTCATTGC-3 . The thermal cycle parameters used were: 2 min at 50 ◦ C, 2 min at 95 ◦ C, 40 cycles of 15 s at 95 ◦ C and 30 s at 60 ◦ C. All the samples were analyzed in triplicate and the mean value of these triplicate measurements were used for the calculations of the mRNA expression. The housekeeping gene -actin was used as an internal standard. Relative expression was calculated by using a modified comparative cycle threshold (UK) method.
40
20
0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL) Fig. 1 – Percentage of female and male zebrafish after exposed to triadimefon from 24 h post fertilization to 120 days post fertilization. Asterisk indicates significantly different proportion of males from the control (*P < 0.05; **P < 0.01).
triadimefon (0.25 and 0.5 g/mL) had decreased length and weight compared with the control fish.
3.2.
Sex ratio and gonad histology
The sex ratio of the groups was presented in Fig. 1. There is a significant bias towards male fish (71%) in the group exposed to 0.5 g/mL triadimefon compared to the control group (52%). None of the gonadal somatic index (GSI) differed significantly between treatments (Table 1), whereas the structure of gonad was different (Fig. 2). All female fish in the control group had ovaries with oocytes in the perinucleolar oocyte stage, cortical alveolus stage and vitellogenic oocyte stage (Fig. 2A). In the 0.5 g/mL triadimefon treatment, the percent distribution of perinucleolar oocyte stage was 44 ± 11%, which was significantly more than the control group (19 ± 5%) (Figs. 2C and 3). Therefore, we could conclude that the female fish in the 0.5 g/mL triadimefon treatment had immature ovaries. All male fish in the control and treated groups has testes with spermatocyte, spermatogonia and spermatozoa stage. No obvious differences in the testicular structure were observed between the control and triadimefon treated groups (Fig. 2B and D).
3.3.
Sperm quality in F0 males
Sperm concentration and motility revealed no significant differences between exposure treatment (triadimefon −0.125, 0.25 g/mL) and the control. However, the male fish exposed to 0.5 g/mL triadimefon had significantly lower sperm concentration and sperm motility than the control group (Fig. 4).
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Table 1 – Mean weight, length and gonadosomatic index (GSI) zebrafish at 120 days post fertilization in zebrafish exposed to triadimefon (0.125, 0.25, 0.5 g/mL) or solvent control (con). Data are means ± SEM. Sex
Treatment (g/mL)
Weight (g)
Length (cm)
GSI (%)
Male
con 0.125 0.25 0.5
0.24 0.23 0.21 0.19
± ± ± ±
0.01 0.02 0.02** 0.01**
2.57 2.44 2.41 2.30
± ± ± ±
0.04 0.02 0.03* 0.05**
1.06 1.03 1.13 1.01
± ± ± ±
0.14 0.14 0.10 0.12
Female
con 0.125 0.25 0.5
0.29 0.29 0.26 0.23
± ± ± ±
0.01 0.02 0.02 0.01*
2.63 2.50 2.48 2.32
± ± ± ±
0.06 0.07 0.05 0.05**
12.65 11.08 11.40 8.73
± ± ± ±
0.83 1.13 1.08 1.19
∗ ∗∗
Statistically significant difference compared to the control group, P < 0.05. Statistically significant difference compared to the control group, P < 0.01.
3.4.
Plasma sex hormones
T and E2 concentrations were analyzed from plasma for each individual separately. The values for plasma E2 and T were shown in Fig. 5. There were no statistically significant differences in plasma sex levels (E2 and T) exposed to triadimefon.
3.5.
mRNA expression of Vtg1 gene in F0 zebrafish
The mRNA expression of Vtg1 in F0 zebrafish was in Fig. 6. The Vtg1 gene transcription was significantly down-regulated 1.4and 2.9-fold upon exposure to 0.25 and 0.5 g/mL triadimefon, respectively.
3.6. F0 breeding trials and quality of offspring F1 embryos During breeding experiments conducted between 90 and 120 dpf, the total number of egg per female in control was 280. However, zebrafish exposed to 0.25 and 0.5 g/mL triadimefon group exhibited a significant reduction in the total number of eggs and spawning per female (Fig. 7). Especially, the female exposed to 0.5 g/mL triadimefon group almost had no spawning, the total number per female was only 4 eggs. There were no malformations observed in the offspring F1 embryos until 120 hpf. No significant difference in the fertility, hatching rate and mortality of zebrafish offspring F1 embryos were observed between the control and lower levels
Fig. 2 – Representative gonadal sections of 120 days post fertilization zebrafish. (A) Mature ovary of control female zebrafish; (B) normal testis of control male zebrafish; (C) immature ovary of 0.5 g/mL triadimefon-treated zebrafish; (D) normal testis of 0.5 g/mL triadimefon-treated zebrafish. Poc, perinucleolar oocyte; Coc, cortical alveolus stage; Voc, vitellogenic oocyte; Sc, spermatocyte; Sg, spermatogonia; Sp, spermatozoa.
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**
(A) 140 Sperm concentration (106/mL)
Percentage of perinucleolar oocyte (%)
60
40
20
0 0
0.125
0.25
120 *
100
80
60
0.5
con
Concentration of triadimefon (µg/mL)
0.125
0.25
0.5
Concentration of triadimefon (µg/mL)
of triadimefon treatment group (0.125 and 0.25 g/mL) (Fig. 8). However, zebrafish exposed to higher level of triadimefon (0.5 g/mL) exhibited a significant reduction of the fertilization rate, only 12% embryos could be fertilized compared to the control (98%). And the fertilized embryos could not be hatched, and was finally totally dead at 120 hpf.
4.
Discussion
In the present study, we examined the effects on sexual development in zebrafish after full life-cycle exposure to triadimefon. The chosen concentrations of triadimefon (0.125, 0.25 and 0.5 g/mL) were environmental levels according to the previous studies (Vincelli, 2004; Watschke et al., 2000). Very limited information was available concerning the effect of triadimefon on the growth of animals. Triadimefon was confirmed to suppress the growth of rodents (Goetz et al., 2007; Rockett et al., 2006). In the present study, one of the most pronounced effects of triadimefon was the decreased body length and body weight, which had not been found in fish previously. The reduction of body weight and body length might be due to the effect of xenoestrogens on vitellogenesis synthesis. Vitellogenesis was demonstrated to compromise growth in fish by diverting energy stores as well as producing a burden of protein for the kidneys (Herman and Kincaid, 1988). Similar growth suppression was also observed in zebrafish exposed to EE2 (Xu et al., 2008). Numerous previous studies have reported that Vtg induction can be a sensitive biomarker for endocrine disruptor in zebrafish (Andersen et al., 2004; Christianson-Heiska et al., 2008; Liu et al., 2010). When exposed to xeno-estrogens or estrogens, e.g., EE2 . Significant Vtg induction in an oestrogenresponsive transgenic zebrafish was occurred following 30-day exposure from fertilization at 3 and 10 ng/L EE2 (Bogers et al., 2006). At 28 dph, Vtg mRNA expression level was elevated
(B) 60
Sperm motility (%)
Fig. 3 – Percentage of perinucleolar oocyte in female zebrafish ovaries after exposed to triadimefon from 24 h post fertilization to 120 days post fertilization. Asterisk indicates significantly different proportion of males from the control (*P < 0.05; **P < 0.01).
40
*
**
0.25
0.5
20
0 con
0.125
Concentration of triadimefon (µg/mL) Fig. 4 – Sperm quality parameters in zebrafish males at 120 days post fertilization after exposed to triadimefon. (A) Sperm concentration. (B) Sperm motility. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
at 10 ng/L EE2 (Xu et al., 2008). In the present study, significantly Vtg gene transcription was down-regulated exposed to triadimefon. A previous study showed that 8:2 flurotelomer alcohol exposure to zebrafish resulted in decreased Vtg gene transcription in female fish (Liu et al., 2010). A significant Vtg decreased was also observed in both female and male zebrafish after exposure to prochloraz (202 g/L) (Kinnberg et al., 2007). These effects were similar to the effects observed in the present study. The decreased Vtg mRNA transcription could be explained by the aromatase inhibiting effect of triadimefon. Exposure of zebrafish to 0.5 g/mL triadimefon from 24 h post fertilization to 120 days post fertilization resulted in a significantly increased proportion of males compared to the control group. This effect was consistent with aromatase inhibition (Scholz and Kluver, 2009). If the balance between estrogens and androgens was essential for the
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(A)
350
Total number of eggs/female
300 250 200
**
150 100 50 **
0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL)
Fig. 5 – Serum sex hormone concentration (ng/mL) in zebrafish at 120 days post fertilization after exposed to triadimefon. (A) testosterone (T) and (B) 17-estradiol (E2).
Total number of spawing/female
(B)
1.4 1.2 1.0 0.8
* 0.6 0.4
**
0.2 0.0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL) sexual differentiation of fish, inhibition of the aromatase, and thus inhibition of oestrogen synthesis, could balance the sex towards males. A number of studies support this presumption. Kinnberg et al. reported that male phenotype was also observed in zebrafish exposed to aromatase inhibitor
Fig. 7 – Total number of eggs (A) and spawning (B) per female zebrafish in breeding trials conducted between 90 and 120 days post fertilization. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
Relative Vtg1 mRNA expression
1.2
1.0 **
0.8
0.6 **
0.4
0.2
0.0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL) Fig. 6 – The gene expressions of Vtg1 were determined by real-time PCR at 120 days post fertilization after exposure to triadimefon. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
prochloraz between 24 h post fertilization and 60 days post fertilization (Kinnberg et al., 2007). Application of aromatase inhibitor fadrozole (500 g/g of food) between 35 and 71 days post fertilization resulted in 100% masculinization (Fenske and Segner, 2004). In fish exposed to aromatase inhibitor fadrozole, the percentage of females declined from 20 to 60 days post hatch (Andersen et al., 2004). In the present study, histological assessment revealed that the percent distribution of perinucleolar oocyte stage in the female fish (triadimefon −0.5 g/mL) was significantly more than the control group. This effect was also consistent with aromatase inhibition. Zebrafish exposed to 202 g/L the aromatase inhibitor prochloraz showed the incidence of the stages of the gonads (Kinnberg et al., 2007). Female fathead minnows exposed for 21 days to fadrozole showed a decrease in mature oocytes (Ankley et al., 2002). However, no difference in the testis was observed between the control and treated groups in the present study. Male fish exposed to 0.5 g/mL triadimefon had lower sperm
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(A) 120
Fertility (%)
100
80
60
40 **
20
0 1
2
3
4
Concentration of triadimefon (µg/mL) (B) 100
Hatching rate (%)
80
60
40
20 ** 0 0
(C)
0.125
0.25
Concentration of triadimefo n ( µ g/mL )
120
*
100
Mortality (%)
0.5
80
60
40
20
0 con
0.125
0.25
0.5
Concentration of triadimefon ( µ g/mL )
Fig. 8 – Percentage of fertility (A), hatching rate (B) and mortality (C) of zebrafish offspring F1 embryos. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
aromatase inhibitor, tributyltin, has the lower sperm motility (McAllister and Kime, 2003). These effects were similar to the effects observed in the present study. The reduction in sperm concentration and motility in male zebrafish could be consistent with the aromatase inhibiting effect of triadimefon. F0 breeding studies revealed significant reproductive dysfunctions in 0.25 and 0.5 g/mL triadimefon-treated groups. This was the first report in fish with reduced eggs and spawning after exposure to triadimefon. Reproductive success is a very complicated process and it might be influenced by multiple factors (Nash et al., 2004). In zebrafish, the courtship behavioural consisted essentially of abrupt turns and an elliptical pattern by male around the female, and it was found that egg production in female zebrafish was triggered by interaction with the male (Darrow and Harris, 2004). In recent years, many studies have been found that the reproductive success of fish has been impaired after exposed to EDCs (Nash et al., 2004; Soares et al., 2009; Xu et al., 2008). The sperm examination of this study revealed that triadimefon decreased the sperm mobility and sperm concentration, which might therefore impair the fertility of zebrafish during spawning. Similar effects were reported in female minnow exposed to EE2 for up to 59 days (Zillioux et al., 2001). In addition, the reduced reproductive success was probably due to the disrupted gonad development, which was previously observed in zebrafish exposed to oestrogen (Coe et al., 2010; Hill and Janz, 2003). In the test for quality of offspring, parental full life-cycle exposure to triadimefon (0.5 g/mL) resulted in an increased in egg mortality. In accordance with the results of egg mortality, an increased in abnormal egg development (fertility and hatching rate) in the highest triadimefon exposure concentration was observed. Since successful development and hatching of embryos were dependent upon the quality of gametes as well as nutritional content of eggs, it also indicated the reproductive impairment by triadimefon treatment. In summary, this study demonstrated that life cycle exposure of zebrafish to the environmental levels of triadimefon severely impaired the sexual development. For instance, exposure of zebrafish to triadimefon delayed sexual maturation, skewed sex ratio towards male, caused reduction of Vtg, these responses were consistent with the aromatase inhibiting effect of triadimefon. Breeding experiments conducted from 90 to 120 dpf in exposed males and females revealed significant reductions in production eggs, spawning, hatchability and fertility, suggesting impairment in breeding success.
Conflict of interest statement concentration and sperm motility. Usually, assessment of sperm concentration and motility provided a valid estimate of the quality of sperm for fertilization in fish (Kime et al., 2001). Hara suggested that nonylphenol caused a reduction in sperm motility in teleost fish in short period between ejaculation and fertilization (Hara et al., 2007). Nonylphenol affected the structure of testis in male platyfish Xiphophorus maculatus, an increase in the amount of hypertrophied sertoli, which was related to the quality of sperm (Kinnberg et al., 2000). A previous study showed that zebrafish exposed to the
Nothing declared.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etap. 2013.11.007.
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references Andersen, L., Kinnberg, K., Holbech, H., Korsgaard, B., Bjerregaard, P., 2004. Evaluation of a 40 day assay for testing endocrine disrupters: effects of an anti-estrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio). Fish Physiol. Biochem. 30, 257–266. Ankley, G.T., Johnson, R.D., 2004. Small fish models for identifying and assessing the effects of endocrine-disrupting chemicals. ILAR J. 45, 469–483. Ankley, G.T., Kahl, M.D., Jensen, K.M., Hornung, M.W., Korte, J.J., Makynen, E.A., Leino, R.L., 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicol. Sci. 67, 121–130. Bogers, R., Mutsaerds, E., Druke, J., De Roode, D.F., Murk, A.J., Van der Burg, B., Legler, J., 2006. Estrogenic endpoints in fish early life-stage tests: luciferase and vitellogenin induction in estrogen-responsive transgenic zebrafish. Environ. Toxicol. Chem. 25, 241–247. Christianson-Heiska, I.L., Haavisto, T., Paranko, J., Bergelin, E., Isomaa, B., 2008. Effects of the wood extractives dehydroabietic acid and betulinol on reproductive physiology of zebrafish (Danio rerio) – a two-generation study. Aquat. Toxicol. 86, 388–396. Coe, T.S., Soffker, M.K., Filby, A.L., Hodgson, D., Tyler, C.R., 2010. Impacts of early life exposure to estrogen on subsequent breeding behavior and reproductive success in zebrafish. Environ. Sci. Technol. 44, 6481–6487. Darrow, K.O., Harris, W.A., 2004. Characterization and development of courtship in zebrafish, Danio rerio. Zebrafish 1, 40–45. EPA, 2006. Reregistration Eligibility Decision: Triadimefon and Tolerance Reassessment for Traidmimenol. US EPA. Fenske, M., Segner, H., 2004. Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquat. Toxicol. 67, 105–126. Georgopapadakou, N.H., Walsh, T.J., 1996. Antifungal agents: chemotherapeutic targets and immunologic strategies. Antimicrob. Agents Chemother. 40, 279–291. Goetz, A.K., Ren, H.Z., Schmid, J.E., Blystone, C.R., Thillainadarajah, I., Best, D.S., Nichols, H.P., Strader, L.F., Wolf, D.C., Narotsky, M.G., Rockett, J.C., Dix, D.J., 2007. Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat. Toxicol. Sci. 95, 227–239. Hallare, A., Nagel, K., Kohler, H.R., Triebskorn, R., 2006. Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos. Ecotoxicol. Environ. Saf. 63, 378–388. Hara, Y., Strussmann, C.A., Hashimoto, S., 2007. Assessment of short-term exposure to nonylphenol in Japanese medaka using sperm velocity and frequency of motile sperm. Arch. Environ. Contam. Toxicol. 53, 406–410. Herman, R.L., Kincaid, H.L., 1988. Pathological effects of orally-administered estradiol to rainbow-trout. Aquaculture 72, 165–172. Hill, R.L., Janz, D.M., 2003. Developmental estrogenic exposure in zebrafish (Danio rerio). I: Effects on sex ratio and breeding success. Aquat. Toxicol. 63, 417–429. Kime, D.E., Van Look, K.J.W., McAllister, B.G., Huyskens, G., Rurangwa, E., Ollevier, F., 2001. Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 130, 425–433. Kinnberg, K., Holbech, H., Petersen, G.I., Bjerregaard, P., 2007. Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio). Comp. Biochem. Phys. C 145, 165–170.
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Kinnberg, K., Korsgaard, B., Bjerregaard, P., Jespersen, A., 2000. Effects of nonylphenol and 17 beta-estradiol on vitellogenin synthesis and testis morphology in male platyfish Xiphophorus maculatus. J. Exp. Biol. 203, 171–181. Liu, C.S., Deng, J., Yu, L.Q., Ramesh, M., Zhou, B.S., 2010. Endocrine disruption and reproductive impairment in zebrafish by exposure to 8:2 fluorotelomer alcohol. Aquat. Toxicol. 96, 70–76. Martyniuk, C.J., Gerrie, E.R., Popesku, J.T., Ekker, M., Trudeau, V.L., 2007. Microarray analysis in the zebrafish (Danio rerio) liver and telencephalon after exposure to low concentration of 17 alpha-ethinylestradiol. Aquat. Toxicol. 84, 38–49. McAllister, B.G., Kime, D.E., 2003. Early life exposure to environmental levels of the aromatase inhibitor tributyltin causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio). Aquat. Toxicol. 65, 309–316. Nash, J.P., Kime, D.E., Van der Ven, L.T.M., Wester, P.W., Brion, F., Maack, G., Stahlschmidt-Allner, P., Tyler, C.R., 2004. Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish. Environ. Health Perspect. 112, 1725–1733. Rasmussen, T.H., Korsgaard, B., 2004. Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout (Zoarces viviparus). Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 139, 1–10. Rockett, J.C., Narotsky, M.G., Thompson, K.E., Thillainadarajah, I., Blystone, C.R., Goetz, A.K., Ren, H., Best, D.S., Murrell, R.N., Nichols, H.P., Schmid, J.E., Wolf, D.C., Dix, D.J., 2006. Effect of conazole fungicides on reproductive development in the female rat. Reprod. Toxicol. 22, 647–658. Scholz, S., Kluver, N., 2009. Effects of endocrine disrupters on sexual, gonadal development in fish. Sex. Dev. 3, 136–151. Semenza, J.C., Tolbert, P.E., Rubin, C.H., Guillette, L.J., Jackson, R.J., 1997. Reproductive toxins and alligator abnormalities at Lake Apopka, Florida. Environ. Health Perspect. 105, 1030–1032. Soares, J., Coimbra, A.M., Reis-Henriques, M.A., Monteiro, N.M., Vieira, M.N., Oliveira, J.M.A., Guedes-Dias, P., Fontainhas-Fernandes, A., Parra, S.S., Carvalho, A.P., Castro, L.F.C., Santos, M.M., 2009. Disruption of zebrafish (Danio rerio) embryonic development after full life-cycle parental exposure to low levels of ethinylestradiol. Aquat. Toxicol. 95, 330–338. Sofikitis, N., Giotitsas, N., Tsounapi, P., Baltogiannis, D., Giannakis, D., Pardalidis, N., 2008. Hormonal regulation of spermatogenesis and spermiogenesis. J. Steroid Biochem. 109, 323–330. UK PAN, 2006. A catalogue of lists of pesticides identifying those associated with particularly harmful health or environmental impacts. Pesticide Action Network, UK. Vincelli, P., 2004. Simulations of fungicide runoff following applications for turfgrass disease control. Plant Dis. 88, 391–396. Watschke, T.L., Mumma, R.O., Linde, D.T., Borger, J.A., Harrison, S.A., 2000. Surface runoff of selected pesticides applied to turfgrasses. Fate Manage. Turfgrass Chem. 743, 94–105. Xu, H., Yang, J., Wang, Y.X., Jiang, Q., Chen, H., Song, H.Y., 2008. Exposure to 17 alpha-ethynylestradiol impairs reproductive functions of both male and female zebrafish (Danio rerio). Aquat. Toxicol. 88, 1–8. Zarn, J.A., Bruschweiler, B.J., Schlatter, J.R., 2003. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. Environ. Health Perspect. 111, 255–261. Zillioux, E.J., Johnson, I.C., Kiparissis, Y., Metcalfe, C.D., Wheat, J.V., Ward, S.G., Liu, H., 2001. The sheepshead minnow as an in vivo model for endocrine disruption in marine teleosts: a partial life-cycle test with 17 alpha-ethynylestradiol. Environ. Toxicol. Chem. 20, 1968–1978.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Disruption of zebrafish (Danio rerio) sexual development after full life-cycle exposure to environmental levels of triadimefon Shao-ying Liu a , Quan Jin a , Xi-hui Huang a , Guo-nian Zhu b,∗ a
Laboratory of Chemistry and Physics, Hangzhou Center for Disease Control and Prevention, Hangzhou 310021, PR China b Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
In this study, zebrafish was exposed to environmental levels of triadimefon (0.125, 0.25,
Received 27 August 2013
0.5 g/mL) from 24 h post fertilization to 120 days post fertilization. Several endpoints that
Received in revised form
related to reproductive function were evaluated. It was found that the body length, body
31 October 2013
weight and vitellogenin transcription were significantly reduced for fish exposed to tri-
Accepted 7 November 2013
adimefon. Histological examination showed that the sex ratio of fish skewed to male and
Available online 1 December 2013
female exposed to 0.5 g/mL triadimefon had immature ovary. The breeding success, as determined from data on egg production and spawning, was reduced in fish exposed to
Keywords:
0.25 g/mL triadimefon. In the offspring, the reduced egg fertility, hatching rate and sur-
Triadimefon
vival were observed in eggs exposed to 0.5 g/mL triadimefon. These findings indicated
Zebrafish
that triadimefon had the potential to adversely affect the sexual development and breeding
Sexual development
success through the multiple endocrine actions.
Breeding success
© 2014 Elsevier B.V. All rights reserved.
Endocrine disrupting compounds
1.
Introduction
There are increasing evidences that a number of environmental substances have the potential to disrupt the endocrine systems of exposed organisms. Many of these socalled endocrine disrupting compounds (EDCs) interfere with the sexual development and reproduction (Rasmussen and Korsgaard, 2004). The potential risk posed by EDCs for human and wildlife has instigated efforts to establish regulatory programmes for EDC hazard assessment. Since the aquatic environment is a major sink for EDCs, a particular attention is given to the development and validation of testing approaches using fish. Zebrafish (Danio rerio) is under consideration as
a test species. Zebrafish develops from the fertilized egg to mature adults within a few months and is therefore perform full life-cycle tests with a variety of developmental and reproductive endpoints that might be affected by EDCs (Ankley and Johnson, 2004). Chemicals may adversely influence endocrine function via several pathways. For example, substances that affect the biosynthesis and metabolism of sex steroids can disturb function of the hypothalamic–pituitary–gonadal axis. In fish, the hormonal balance between estrogens and androgens appears to be an important factor in the course of sexual differentiation. For example, exposure of zebrafish to estrogens during early development has been shown to change the sex ratio towards more females or more males, respectively
∗ Corresponding author at: Institute of Pesticide and Environmental Toxicology, Zhejiang University, Kaixuan Road 172, Hangzhou, Zhejiang 310029, PR China. Fax: +86 571 8643 0193. E-mail address: [email protected] (G.-n. Zhu). 1382-6689/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2013.11.007
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 468–475
(Scholz and Kluver, 2009; Sofikitis et al., 2008). The balance of the sex hormone depends on the availability and activity of the steroid synthesizing enzymes, and particularly on the CYP19 aromatase, which catalyses the conversion of androgens to estrogens. The antifungal action of triadimefon is based on the inhibition of the cytochrome P450-dependent 14␣-demethylase required in the biosynthesis of ergosterol, a fungal cell membrane (Georgopapadakou and Walsh, 1996). However, triadimefon also modulates the activity of other cytochrome P450-dependent enzymes, including enzymes involved in vertebrate biosynthesis of steroids as for example CYP19 aromatase (Zarn et al., 2003). Triadimefon has a potential endocrine disruption effect from reports of European Union (EU) and World Wide Fund for Nature (WWF) in 2006 (UK, 2006). Environmental triadimefon concentrations in water are highly variable, from 0.046 to 0.922 g/mL (Watschke et al., 2000). From other study, the majority of the measured concentrations are below 0.3 g/mL (Vincelli, 2004). Triadimefon has the ability to bioconcentration in fish tissue (EPA, 2006). In the model teleost, zebrafish (Danio rerio), none paper has been conducted to illustrate the impact on reproductive parameters cover the full life-cycle to triadimefon. Therefore, the main aim of the present study was to examine the concentration-dependent effects of zebrafish full life-cycle exposure to low levels of triadimefon on the sexual development. In order to achieve this, zebrafish were exposed to low levels of triadimefon (0.125, 0.25, 0.5 g/mL), from 24 h post fertilization (hpf) to 120 days post fertilization (dpf). During this chronic exposure, some parameters of sexual development, such as gonad histological examination and sex ratios were investigated.
2.
Materials and methods
2.1.
Chemicals
Triadimefon (96%) was purchased from Bayer Crop Science. The stock solution of 5 mg/mL triadimefon was prepared in acetone and stored in darkness. Test solutions were prepared by dilution of the stock solution with the test water (aerated tap water), hardness (86 mg/L), pH value (6.5–7.5). The final concentration of acetone was not exceeding 0.01%, which was inactive to zebrafish (Hallare et al., 2006).
2.2.
Experimental animals and embryos collections
Adult wild-type zebrafish were maintained at 28 ± 1 ◦ C under a light/dark cycle of 14 h:10 h. Embryos were obtained from healthy adult fish with the sex ratio of 1:1. Spawning was induced in the next morning when the light was turned on. Fertilized and normal embryos were collected within 0.5 h of spawning and rinsed in the test water twice.
2.3.
Full life-cycle exposure of parental (F0) generation
About 50 embryos of 24 h post fertilization (hpf) were exposed to triadimefon (0.125, 0.25, 0.5 g/mL) and solvent control (0.01%) in 1 L water tanks. Triplicate exposures were performed
469
for each test chemical concentration and solvent control. Every 48 h from 24 hpf to 120 dpf, a 100% water change was performed and fry were transferred to clean, acetone rinsed tanks. Fresh acetone or test chemicals were added at the time of the water change. Mortality and any abnormalities in appearance of surviving fish were observed and recorded daily over the 120-day exposure period. Dead fish were removed and discarded according to animal care protocols. At 40 dpf, 5 fish from each treatment and control were randomly selected for Vtg determination and there were three replications for each exposure concentration. Starting at exposure day 90 and concluding on exposure day 120, breeding trials were carried out to estimate the breeding success. At study termination, the fish from all replicates of a given treatment were pooled. The fish from each pooled group were selected for measurement of lengths, weights, sex ratio, sperm examination and gonad histology.
2.4. Physiological and biochemical indexes of parental (F0) generation 2.4.1.
Histological examination
At 120 dpf, the fish from all replicates of a given treatment group were pooled. The fish from each pooled group were randomly selected for measurement of weights and lengths. The gonads were get out and the gonadal somatic index (GSI) (gonad weight(g)/body weight(g) × 100) and the sex ratios were calculated. Gonads were fixed with 2.5% glutaraldehyde in phosphate buffer and then embedded in paraffin and 2 m sectioned were cut longitudinally through the gonad. The sections were stained with 0.1% methylthionine chloride and examined by light microscopy.
2.4.2.
Plasma sex hormones
At 120 dpf, blood samples from individual were collected from the caudal vein. There were three replicates for the control and the exposure treatments. 17-estradiol (E2) and testosterone (T) were detected using hormone detection kits (RD Company, USA), following the manufacturer’s instructions. The assay detection limit for T was 0.216 ng/mL and 0.100 ng/mL for E2.
2.4.3.
Sperm quality parameters in F0 males
At 120 dpf, the sperm from the male fish in each treatment were collected and diluted with HBSS 300 to achieve sperm suspension. Sperm concentration and motility were determined via a computer-assisted sperm analysis system (CASA, IVOS version 12.0, Hamilton Thorne Bioscience, Beverly, MA, USA). Five to six fields were analyzed and each of the sperm samples was tested in triplicate.
2.4.4.
Vtg mRNA expression
In this study, Vtg mRNA expression of zebrafish (38 dpf) was determined using quantitative real-time PCR. Total RNA was extracted from 5 homogenized fish by using Trizol Reagent (Invitrogen, Carlsbad CA, USA) according to the manufacturer’s instructions. To remove the genomic DNA contamination, the total RNA mixture was treated with RNaefree DNase I (Promega Madison, WI, USA) and then purified. Approximately 2 g RNA was used for cDNA Synthesis with M-MLV RTase (Takara Biochemicals, Dalian, China) according
470
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2.5.
F0 breeding trials and quality of offspring F1
During breeding trials conducted between 90 and 120 dpf, six randomly selected fish from each exposure concentration were placed into a breeding chamber in triadimefon (0.125, 0.25, 0.5 g/mL) and solvent control (0.01%) continuously. Three replicates of each exposure concentration were used for each trial. Fish were bred for 20 consecutive days with eggs being collected each day. The total number of eggs and spawning per female were used to assess the reproductive ability of parental (F0) generation. Then F1 embryos were maintained in the clean water until 120 hpf, the embryo fertility, mortality, hatching rate and malformation were monitored to assess the quality of offspring.
2.6.
Statistical analysis
One-way ANOVA was applied to calculate statistical significance followed by Dunnett’s test as a post hoc test to independently compare each exposure group to the control group. The LSD test was used as a post hoc test for multiple comparisons between groups. *P < 0.05 and **P < 0.01 were regarded as statistically significant using SPSS 16.0 (SPSS, Chicago, IL, USA). Data were presented as means ± standard error (Semenza et al., 1997).
3.
Results
3.1.
Survival and growth at 120 dpf
The cumulative mortalities, from 24 hpf to 120 dpf, varied between 64% and 80% and no differences were observed between control and triadimefon exposure groups (data not shown). These mortality rates were within the normal expected values for zebrafish (Soares et al., 2009). The weight and length of female fish exposed to 0.5 g/mL triadimefon were significantly lower than those of the control at 120 dpf (Table 1). Meanwhile, male fish exposed to
Male
Female *
100
80
60
%
to manufacturer’s description. The product from the reverse transcription was diluted 25 times and 5 L of each diluted sample, corresponding to 10 ng of the reverse transcribed RNA, was used for the real-time PCR. Quantitative real-time polymerase chain reaction (PCR) was performed by using the SYBR Green PCR kit (Takara Biochemicals, Dalian, China). Primers for Vtg1 and housekeeping gene (-actin) were performed as described previously by Martyniuk et al. (2007): Vtg1 upstream primer 5 -TCCATTGCTGAAAACGACAA-3 ; downstream primer 5 -GGCAGTCTCTCCCATTCCAC-3 ; actin upstream primer 5 -CGAGCAGGAGATGGGAACC-3 ; and -actin downstream primer 5 -CAACGGAAACGCTCATTGC-3 . The thermal cycle parameters used were: 2 min at 50 ◦ C, 2 min at 95 ◦ C, 40 cycles of 15 s at 95 ◦ C and 30 s at 60 ◦ C. All the samples were analyzed in triplicate and the mean value of these triplicate measurements were used for the calculations of the mRNA expression. The housekeeping gene -actin was used as an internal standard. Relative expression was calculated by using a modified comparative cycle threshold (UK) method.
40
20
0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL) Fig. 1 – Percentage of female and male zebrafish after exposed to triadimefon from 24 h post fertilization to 120 days post fertilization. Asterisk indicates significantly different proportion of males from the control (*P < 0.05; **P < 0.01).
triadimefon (0.25 and 0.5 g/mL) had decreased length and weight compared with the control fish.
3.2.
Sex ratio and gonad histology
The sex ratio of the groups was presented in Fig. 1. There is a significant bias towards male fish (71%) in the group exposed to 0.5 g/mL triadimefon compared to the control group (52%). None of the gonadal somatic index (GSI) differed significantly between treatments (Table 1), whereas the structure of gonad was different (Fig. 2). All female fish in the control group had ovaries with oocytes in the perinucleolar oocyte stage, cortical alveolus stage and vitellogenic oocyte stage (Fig. 2A). In the 0.5 g/mL triadimefon treatment, the percent distribution of perinucleolar oocyte stage was 44 ± 11%, which was significantly more than the control group (19 ± 5%) (Figs. 2C and 3). Therefore, we could conclude that the female fish in the 0.5 g/mL triadimefon treatment had immature ovaries. All male fish in the control and treated groups has testes with spermatocyte, spermatogonia and spermatozoa stage. No obvious differences in the testicular structure were observed between the control and triadimefon treated groups (Fig. 2B and D).
3.3.
Sperm quality in F0 males
Sperm concentration and motility revealed no significant differences between exposure treatment (triadimefon −0.125, 0.25 g/mL) and the control. However, the male fish exposed to 0.5 g/mL triadimefon had significantly lower sperm concentration and sperm motility than the control group (Fig. 4).
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Table 1 – Mean weight, length and gonadosomatic index (GSI) zebrafish at 120 days post fertilization in zebrafish exposed to triadimefon (0.125, 0.25, 0.5 g/mL) or solvent control (con). Data are means ± SEM. Sex
Treatment (g/mL)
Weight (g)
Length (cm)
GSI (%)
Male
con 0.125 0.25 0.5
0.24 0.23 0.21 0.19
± ± ± ±
0.01 0.02 0.02** 0.01**
2.57 2.44 2.41 2.30
± ± ± ±
0.04 0.02 0.03* 0.05**
1.06 1.03 1.13 1.01
± ± ± ±
0.14 0.14 0.10 0.12
Female
con 0.125 0.25 0.5
0.29 0.29 0.26 0.23
± ± ± ±
0.01 0.02 0.02 0.01*
2.63 2.50 2.48 2.32
± ± ± ±
0.06 0.07 0.05 0.05**
12.65 11.08 11.40 8.73
± ± ± ±
0.83 1.13 1.08 1.19
∗ ∗∗
Statistically significant difference compared to the control group, P < 0.05. Statistically significant difference compared to the control group, P < 0.01.
3.4.
Plasma sex hormones
T and E2 concentrations were analyzed from plasma for each individual separately. The values for plasma E2 and T were shown in Fig. 5. There were no statistically significant differences in plasma sex levels (E2 and T) exposed to triadimefon.
3.5.
mRNA expression of Vtg1 gene in F0 zebrafish
The mRNA expression of Vtg1 in F0 zebrafish was in Fig. 6. The Vtg1 gene transcription was significantly down-regulated 1.4and 2.9-fold upon exposure to 0.25 and 0.5 g/mL triadimefon, respectively.
3.6. F0 breeding trials and quality of offspring F1 embryos During breeding experiments conducted between 90 and 120 dpf, the total number of egg per female in control was 280. However, zebrafish exposed to 0.25 and 0.5 g/mL triadimefon group exhibited a significant reduction in the total number of eggs and spawning per female (Fig. 7). Especially, the female exposed to 0.5 g/mL triadimefon group almost had no spawning, the total number per female was only 4 eggs. There were no malformations observed in the offspring F1 embryos until 120 hpf. No significant difference in the fertility, hatching rate and mortality of zebrafish offspring F1 embryos were observed between the control and lower levels
Fig. 2 – Representative gonadal sections of 120 days post fertilization zebrafish. (A) Mature ovary of control female zebrafish; (B) normal testis of control male zebrafish; (C) immature ovary of 0.5 g/mL triadimefon-treated zebrafish; (D) normal testis of 0.5 g/mL triadimefon-treated zebrafish. Poc, perinucleolar oocyte; Coc, cortical alveolus stage; Voc, vitellogenic oocyte; Sc, spermatocyte; Sg, spermatogonia; Sp, spermatozoa.
472
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**
(A) 140 Sperm concentration (106/mL)
Percentage of perinucleolar oocyte (%)
60
40
20
0 0
0.125
0.25
120 *
100
80
60
0.5
con
Concentration of triadimefon (µg/mL)
0.125
0.25
0.5
Concentration of triadimefon (µg/mL)
of triadimefon treatment group (0.125 and 0.25 g/mL) (Fig. 8). However, zebrafish exposed to higher level of triadimefon (0.5 g/mL) exhibited a significant reduction of the fertilization rate, only 12% embryos could be fertilized compared to the control (98%). And the fertilized embryos could not be hatched, and was finally totally dead at 120 hpf.
4.
Discussion
In the present study, we examined the effects on sexual development in zebrafish after full life-cycle exposure to triadimefon. The chosen concentrations of triadimefon (0.125, 0.25 and 0.5 g/mL) were environmental levels according to the previous studies (Vincelli, 2004; Watschke et al., 2000). Very limited information was available concerning the effect of triadimefon on the growth of animals. Triadimefon was confirmed to suppress the growth of rodents (Goetz et al., 2007; Rockett et al., 2006). In the present study, one of the most pronounced effects of triadimefon was the decreased body length and body weight, which had not been found in fish previously. The reduction of body weight and body length might be due to the effect of xenoestrogens on vitellogenesis synthesis. Vitellogenesis was demonstrated to compromise growth in fish by diverting energy stores as well as producing a burden of protein for the kidneys (Herman and Kincaid, 1988). Similar growth suppression was also observed in zebrafish exposed to EE2 (Xu et al., 2008). Numerous previous studies have reported that Vtg induction can be a sensitive biomarker for endocrine disruptor in zebrafish (Andersen et al., 2004; Christianson-Heiska et al., 2008; Liu et al., 2010). When exposed to xeno-estrogens or estrogens, e.g., EE2 . Significant Vtg induction in an oestrogenresponsive transgenic zebrafish was occurred following 30-day exposure from fertilization at 3 and 10 ng/L EE2 (Bogers et al., 2006). At 28 dph, Vtg mRNA expression level was elevated
(B) 60
Sperm motility (%)
Fig. 3 – Percentage of perinucleolar oocyte in female zebrafish ovaries after exposed to triadimefon from 24 h post fertilization to 120 days post fertilization. Asterisk indicates significantly different proportion of males from the control (*P < 0.05; **P < 0.01).
40
*
**
0.25
0.5
20
0 con
0.125
Concentration of triadimefon (µg/mL) Fig. 4 – Sperm quality parameters in zebrafish males at 120 days post fertilization after exposed to triadimefon. (A) Sperm concentration. (B) Sperm motility. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
at 10 ng/L EE2 (Xu et al., 2008). In the present study, significantly Vtg gene transcription was down-regulated exposed to triadimefon. A previous study showed that 8:2 flurotelomer alcohol exposure to zebrafish resulted in decreased Vtg gene transcription in female fish (Liu et al., 2010). A significant Vtg decreased was also observed in both female and male zebrafish after exposure to prochloraz (202 g/L) (Kinnberg et al., 2007). These effects were similar to the effects observed in the present study. The decreased Vtg mRNA transcription could be explained by the aromatase inhibiting effect of triadimefon. Exposure of zebrafish to 0.5 g/mL triadimefon from 24 h post fertilization to 120 days post fertilization resulted in a significantly increased proportion of males compared to the control group. This effect was consistent with aromatase inhibition (Scholz and Kluver, 2009). If the balance between estrogens and androgens was essential for the
473
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 468–475
(A)
350
Total number of eggs/female
300 250 200
**
150 100 50 **
0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL)
Fig. 5 – Serum sex hormone concentration (ng/mL) in zebrafish at 120 days post fertilization after exposed to triadimefon. (A) testosterone (T) and (B) 17-estradiol (E2).
Total number of spawing/female
(B)
1.4 1.2 1.0 0.8
* 0.6 0.4
**
0.2 0.0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL) sexual differentiation of fish, inhibition of the aromatase, and thus inhibition of oestrogen synthesis, could balance the sex towards males. A number of studies support this presumption. Kinnberg et al. reported that male phenotype was also observed in zebrafish exposed to aromatase inhibitor
Fig. 7 – Total number of eggs (A) and spawning (B) per female zebrafish in breeding trials conducted between 90 and 120 days post fertilization. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
Relative Vtg1 mRNA expression
1.2
1.0 **
0.8
0.6 **
0.4
0.2
0.0 0
0.125
0.25
0.5
Concentration of triadimefon (µg/mL) Fig. 6 – The gene expressions of Vtg1 were determined by real-time PCR at 120 days post fertilization after exposure to triadimefon. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
prochloraz between 24 h post fertilization and 60 days post fertilization (Kinnberg et al., 2007). Application of aromatase inhibitor fadrozole (500 g/g of food) between 35 and 71 days post fertilization resulted in 100% masculinization (Fenske and Segner, 2004). In fish exposed to aromatase inhibitor fadrozole, the percentage of females declined from 20 to 60 days post hatch (Andersen et al., 2004). In the present study, histological assessment revealed that the percent distribution of perinucleolar oocyte stage in the female fish (triadimefon −0.5 g/mL) was significantly more than the control group. This effect was also consistent with aromatase inhibition. Zebrafish exposed to 202 g/L the aromatase inhibitor prochloraz showed the incidence of the stages of the gonads (Kinnberg et al., 2007). Female fathead minnows exposed for 21 days to fadrozole showed a decrease in mature oocytes (Ankley et al., 2002). However, no difference in the testis was observed between the control and treated groups in the present study. Male fish exposed to 0.5 g/mL triadimefon had lower sperm
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(A) 120
Fertility (%)
100
80
60
40 **
20
0 1
2
3
4
Concentration of triadimefon (µg/mL) (B) 100
Hatching rate (%)
80
60
40
20 ** 0 0
(C)
0.125
0.25
Concentration of triadimefo n ( µ g/mL )
120
*
100
Mortality (%)
0.5
80
60
40
20
0 con
0.125
0.25
0.5
Concentration of triadimefon ( µ g/mL )
Fig. 8 – Percentage of fertility (A), hatching rate (B) and mortality (C) of zebrafish offspring F1 embryos. Asterisks indicate a statistically significant difference from control (*P < 0.05; **P < 0.01).
aromatase inhibitor, tributyltin, has the lower sperm motility (McAllister and Kime, 2003). These effects were similar to the effects observed in the present study. The reduction in sperm concentration and motility in male zebrafish could be consistent with the aromatase inhibiting effect of triadimefon. F0 breeding studies revealed significant reproductive dysfunctions in 0.25 and 0.5 g/mL triadimefon-treated groups. This was the first report in fish with reduced eggs and spawning after exposure to triadimefon. Reproductive success is a very complicated process and it might be influenced by multiple factors (Nash et al., 2004). In zebrafish, the courtship behavioural consisted essentially of abrupt turns and an elliptical pattern by male around the female, and it was found that egg production in female zebrafish was triggered by interaction with the male (Darrow and Harris, 2004). In recent years, many studies have been found that the reproductive success of fish has been impaired after exposed to EDCs (Nash et al., 2004; Soares et al., 2009; Xu et al., 2008). The sperm examination of this study revealed that triadimefon decreased the sperm mobility and sperm concentration, which might therefore impair the fertility of zebrafish during spawning. Similar effects were reported in female minnow exposed to EE2 for up to 59 days (Zillioux et al., 2001). In addition, the reduced reproductive success was probably due to the disrupted gonad development, which was previously observed in zebrafish exposed to oestrogen (Coe et al., 2010; Hill and Janz, 2003). In the test for quality of offspring, parental full life-cycle exposure to triadimefon (0.5 g/mL) resulted in an increased in egg mortality. In accordance with the results of egg mortality, an increased in abnormal egg development (fertility and hatching rate) in the highest triadimefon exposure concentration was observed. Since successful development and hatching of embryos were dependent upon the quality of gametes as well as nutritional content of eggs, it also indicated the reproductive impairment by triadimefon treatment. In summary, this study demonstrated that life cycle exposure of zebrafish to the environmental levels of triadimefon severely impaired the sexual development. For instance, exposure of zebrafish to triadimefon delayed sexual maturation, skewed sex ratio towards male, caused reduction of Vtg, these responses were consistent with the aromatase inhibiting effect of triadimefon. Breeding experiments conducted from 90 to 120 dpf in exposed males and females revealed significant reductions in production eggs, spawning, hatchability and fertility, suggesting impairment in breeding success.
Conflict of interest statement concentration and sperm motility. Usually, assessment of sperm concentration and motility provided a valid estimate of the quality of sperm for fertilization in fish (Kime et al., 2001). Hara suggested that nonylphenol caused a reduction in sperm motility in teleost fish in short period between ejaculation and fertilization (Hara et al., 2007). Nonylphenol affected the structure of testis in male platyfish Xiphophorus maculatus, an increase in the amount of hypertrophied sertoli, which was related to the quality of sperm (Kinnberg et al., 2000). A previous study showed that zebrafish exposed to the
Nothing declared.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etap. 2013.11.007.
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