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Effects of butachlor on reproduction and hormone levels in adult zebrafish (Danio rerio)
Experimental and Toxicologic Pathology 65 (2013) 205–209
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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Effects of butachlor on reproduction and hormone levels in adult zebrafish (Danio rerio) Juhua Chang a , Shaoying Liu b , Shengli Zhou c , Minghua Wang a,∗∗ , Guonian Zhu b,∗ a Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing 210095, China b Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China c Zhejiang Environmental Monitoring Center, Hangzhou 310015, China
a r t i c l e
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Article history: Received 3 May 2011 Accepted 18 August 2011 Keywords: Butachlor Reproduction Sex steroid Thyroid hormone Vitellogenin Zebrafish
a b s t r a c t Butachlor, a chloracetamide herbicide, is widely used in China. In the present study, paired adult male and female zebrafish (Danio rerio) were exposed to various concentrations of butachlor (0, 25, 50 and 100 g/L) for 30 days, and the effects on reproduction and endocrine disruption were evaluated using fecundity, condition factor (CF), gonadosomatic index (GSI), liver somatic index (LSI), plasma vitellogenin (VTG), sex steroids and thyroid hormone levels as endpoints. Our results showed that the mean fecundity rates were significantly decreased at 50 and 100 g/L butachlor during the 30-day exposure period. At the end of the exposure period, no significant changes were observed in CF and LSI in both females and males, while GSI was significantly reduced in males at 50 and 100 g/L butachlor. At 100 g/L butachlor, plasma testosterone (T) and 17-estradiol (E2) levels were significantly decreased in females, while plasma VTG level was significantly increased in males. Plasma thyroxine (T4) and triiodothyronine (T3) levels were significantly increased at 50 and 100 g/L butachlor in males, and at 100 g/L in females. This work demonstrated that butachlor adversely affected the normal reproductive success of zebrafish, and disrupted the thyroid and sex steroid endocrine systems, which provides the basis for the estimated ecological risk during butachlor exposure. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Man-made pesticides are among the most common sources of environmental pollutants worldwide (Williams, 1996). Butachlor is a chloracetamide herbicide, and is one of the top three herbicides extensively applied to control weeds in rice fields in China (Yu et al., 2003). The herbicide has contaminated river water via the effluents from rice paddy fields (Ohyama et al., 1987). Residues of this herbicide are present in groundwater, including that used in some cases for human consumption (Natarajan, 1993). Butachlor is persistent in agricultural soil and water systems (Yu et al., 2003), posing a potential threat to the agro-ecosystem and human health (Debnath et al., 2002; Sinha et al., 1995). Most experiments investigating the effects of butachlor have focused on genotoxicity to fish, amphibians and rats (Ateeq et al., 2002, 2006; Geng et al., 2005), and some investigations have indicated that butachlor is a suspected carcinogen (Ou et al., 2000). Tomlin reported on the acute toxicity of butachlor to fish, with LC50 (96 h) values ranging from 0.14 to
∗ Corresponding author. Tel.: +86 0571 86971220; fax: +86 0571 86971902. ∗∗ Corresponding author. Tel.: +86 025 84395479; fax: +86 025 84395479. E-mail addresses: [email protected] (M. Wang), [email protected] (G. Zhu). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.08.007
0.52 mg/L, which showed that butachlor was highly toxic to fish (Tomlin, 1994). However, little is known about the reproductive toxicity of butachlor. A short-term fish reproduction assay has been developed for detecting the effects of endocrine-disrupting compounds (EDCs) (Ankley et al., 2001, 2002). A suite of biomarkers is needed to best assess possible disturbances of the endocrine system in wildlife. The use of vitellogenin (VTG) to screen for estrogen exposure in oviparous vertebrates is well-described (Denslow et al., 1999; Harries et al., 2000; Jobling et al., 2003; Korte et al., 2000). Plasma sex steroids (T and E2) have also been used in the study of EDCs in fish (Ankley et al., 2001, 2002). In fish, growth and reproduction are believed to be, at least partly, under the control of the thyroid hormones, thyroxine (T4) and triiodothyronine (T3) (Cyr and Eales, 1996; Power et al., 2001). The zebrafish (Danio rerio) is an attractive model organism for evaluating reproductive toxicity and endocrine-disrupting effects because of its small size, ease of culture, short life cycle and prolific egg production with high fertilization and hatching rates (Segner, 2009). This study was conducted to evaluate the effects of butachlor on reproduction and endocrine disruption in adult zebrafish using a short-term (30 days) assay. In this assay, fecundity was assessed during the exposure period. At the end of the exposure period, this
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J. Chang et al. / Experimental and Toxicologic Pathology 65 (2013) 205–209
study integrated different endpoints such as plasma vitellogenin (VTG), sex steroids (T and E2), and thyroid hormones (T4 and T3) levels, gonadosomatic index (GSI), liver somatic index (LSI) and condition factor (CF) of the zebrafish.
Table 1 Condition factor (CF), gonadosomatic index (GSI) and liver somatic index (LSI) for adult male and female zebrafish after 30 days of exposure to butachlor (n = 15). Index
Sex
Control
CF (%)
♀ ♂ ♀ ♂ ♀ ♂
1.47 1.26 13.10 1.26 2.81 1.59
2. Materials and methods GSI (%)
2.1. Chemicals Butachlor (technical grade AI: 96%) was purchased from Hangzhou Qingfeng Agrochemical Co., Ltd. Butachlor was dissolved in dimethyl sulfoxide (DMSO), and the stock solution (100,000 mg/L) was stored in the dark at 4 ◦ C. MS-222 (3aminobenzoic acid ethyl ester, methanesulfonate salt) was purchased from Sigma (St. Louis, MO, USA). All other chemicals used were analytical grade. 2.2. Test fish and culture conditions Adult zebrafish (D. rerio) were purchased from a local fish shop. The fish were fed with freshly hatched brine shrimp twice a day and were maintained at 28 ± 1 ◦ C and a 14:10 h light:dark photoperiod. Prior to exposure to the herbicide, 100 pairs of zebrafish (one pair/tank) were set up to assess reproductive viability over three weeks, and egg production was recorded daily. Sixty pairs of zebrafish were then selected for butachlor exposure according to fecundity during the three weeks of acclimation. 2.3. Exposure and experimental design Adult zebrafish were exposed to various concentrations (0, 25, 50 and 100 g/L) of butachlor for 30 days, and fifteen pairs of fish were included in the control group and the butachlor-treatment group. One male and one female were set up in a 2 L spawning tank, containing 1.5 L test solution, which was changed daily. Both the exposure and control group received 0.001% (v/v) DMSO. Eight pairs in each treatment were selected to evaluate the effects of butachlor on the fecundity of zebrafish. Two hour after the light had gone on in the morning, spawned eggs were collected and counted from each tank daily. Fecundity rate (as the mean number of eggs per female per day), oviposition (spawning) frequency per female, the mean number of eggs per female per spawning and cumulatively per dose were calculated for the whole reproductive test period (30 days) for each treatment. After 30 days of exposure, all fish were anesthetized with 0.03% MS-222. After measurement of body weight and total length, the gonads and livers were removed and weighed. The gonadosomatic index (GSI = 100·[gonad weight (g)/body weight (g)]), liver somatic index (LSI = 100·[liver weight (g)/body weight (g)]), and condition factor (=100·[body weight (g)/total length3 (cm)]) were calculated. Blood samples were collected from the caudal vein of each fish into heparinized microcapillary tubes, and the blood samples from five fish of the same sex were pooled as one replicate. Three replicates for the control and exposure groups were obtained. After volume measurement, a 5-fold volume of phosphate buffer (10 mM K2 HPO4 , 100 mM KCl, 1 mM EDTA, 1 Mm DTT, pH 7.4) was added to each tube. The samples were immediately centrifuged (10,000 × g, 10 min, 4◦ C), and the obtained plasma samples were stored at −80 ◦ C until analysis.
LSI (%) *
± ± ± ± ± ±
25 g/L 0.17 0.04 1.13 0.19 0.58 0.41
1.57 1.47 10.97 0.85 2.54 1.15
± ± ± ± ± ±
50 g/L 0.07 0.09 0.70 0.20 0.21 0.13
1.73 1.40 10.13 0.80 2.07 1.51
± ± ± ± ± ±
100 g/L 0.11 0.05 0.88 0.08* 0.22 0.24
1.57 1.38 11.39 0.68 1.89 1.40
± ± ± ± ± ±
0.15 0.09 1.31 0.11* 0.24 0.37
Significant difference compared with the control (P < 0.05).
2.5. Statistical analysis Data were statistically analyzed using the SAS program (SAS, 1990) and the Proc GLM procedure was used for variance analyses. Mean separation was conducted using SAS ProcMeans/LSD procedures. The value P < 0.05 was used as the criterion for statistical significance. All data are expressed as mean ± SE. 3. Results 3.1. Effects of butachlor on condition factor, gonadosomatic index and liver somatic index in zebrafish No mortality was observed in any of the treatment groups during the exposure period. Butachlor did not affect condition factor (CF) or liver somatic index (LSI) in females or males in the butachlor-treated groups after 30 days of exposure (Table 1). Reduced gonadosomatic index (GSI) was observed in males in the 50 and 100 g/L butachlor-treated groups compared to the control fish (F = 3.53, P = 0.0236; Table 1). In female fish, no significant differences in the GSI between the control and treatment groups were observed (F = 1.51, P > 0.05; Table 1). 3.2. Effects of butachlor on reproduction in zebrafish Spawning events and egg production were assessed during the 30-day exposure period. Total egg production ranged from 2225 to 7118 across all treatments. Each female produced 278–890 eggs. The mean fecundity rates (eggs/female/day) in the 50 and 100 g/L treatments were 15.46 ± 3.42 and 7.32 ± 2.35, respectively, which were significantly lower than that of the control (27.49 ± 5.89; F = 6.02, P = 0.0027; Fig. 2A). The significant reduction in cumulative eggs produced in the 50 and 100 g/L treatments (Fig. 1) was apparently associated with the reduced number of eggs per spawn
2.4. Plasma hormones and vitellogenin measurement Plasma T, E2, VTG, T4 and T3 levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (RD Chemical, Mountain View, CA, USA), following the manufacturer’s instructions.
Fig. 1. Cumulative number of eggs produced by zebrafish breeding pairs exposed to various concentrations of butachlor for 30 days (n = 8).
J. Chang et al. / Experimental and Toxicologic Pathology 65 (2013) 205–209
Fig. 2. Reproductive parameters of zebrafish breeding pairs exposed to various concentrations of butachlor for 30 days. (A) Eggs per female per day, (B) eggs per female per spawning, and (C) the number of spawning event per female. Data are expressed as the mean ± SE. (n = 8), *significant difference compared with the control (P < 0.05).
(F = 21.06, P < 0.0001; Fig. 2B) and the reduced number of spawning events (F = 6.23, P = 0.0022; Fig. 2C).
3.3. Effects of butachlor on plasma sex steroid and vitellogenin levels in zebrafish The results of the zebrafish plasma sex steroid and vitellogenin analyses are summarized in Fig. 3. After 30 days of exposure, plasma T and E2 levels were significantly decreased in females at 100 g/L butachlor (for T, F = 5.24, P = 0.0271; for E2, F = 2.52, P = 0.1319; Fig. 3A and B), while no significant differences in plasma T and E2 levels in males were observed between the exposure and control groups (for T, F = 0.63, P > 0.05; for E2, F = 1.14, P > 0.05; Fig. 3A and B). Moreover, exposure to 100 g/L butachlor induced a significantly elevated level of VTG in males (F = 13.51, P = 0.0011; Fig. 3C), but did not affect VTG level in females (F = 0.77, P > 0.05; Fig. 3C).
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Fig. 3. Plasma levels of (A) T, (B) E2 and (C) VTG in zebrafish after 30 days of exposure to butachlor. Data are expressed as the mean ± SE. (five fish as a pool, n = 3), *significant difference compared with the control (P < 0.05).
3.4. Effects of butachlor on plasma thyroid hormone levels in zebrafish The results of the zebrafish plasma thyroid hormone analyses are summarized in Fig. 4. At the end of the exposure period, plasma T4 and T3 concentrations in males were significantly increased in the 50 and 100 g/L butachlor-treated groups compared to the control group (for T4, F = 3.26, P = 0.0807; for T3 F = 44.51, P < 0.0001; Fig. 4A and B); In females, plasma T3 and T4 concentrations were significantly increased in the 100 g/L butachlor-treated group compared with the control group (for T4, F = 6.01, P = 0.019; for T3, F = 49.38, P < 0.0001; Fig. 4A and B). 4. Discussion Environmental chemicals that interfere with reproduction in fish have attracted significant attention in recent studies (Ankley et al., 2001, 2002). In the present study, a short-term (30 days) reproduction assay were performed to evaluate the effects of
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Fig. 4. Plasma levels of (A) T4 and (B) T3 in zebrafish after 30 days of exposure to butachlor. Data are expressed as the mean ± SE. (five fish as a pool, n = 3), *significant difference compared with the control (P < 0.05).
butachlor on reproduction and endocrine disruption in adult zebrafish, and ELISA was used to analyze the changes in plasma T, E2, VTG, T4 and T3 levels. It has been proposed that condition factor (CF) can be used as a non-specific measure of physiological fitness in fish health (Anderson et al., 2003), because CF is reflective of changes in food intake, fat deposition, and protein budgets (Smolders et al., 2003). In our study, butachlor did not affect CF in both females and males in all butachlor-treated groups after 30 days exposure. Therefore, the effects of butachlor on reproductive output were clearly sublethal and might be mediated via endocrine disruption. Reproduction is dependent upon the appropriate coordination of the entire hypothalamus–pituitary–gonad (HPG) axis, therefore, chemicals acting at any level of this axis via any mechanism can have adverse effects on reproduction (Brian et al., 2007). Our results indicated that the fecundity rates in the 50 and 100 g/L butachlor-treatments were significantly decreased during the 30-day exposure period. Sex steroids play a crucial role in sex differentiation, sexual maturation, and various female behaviors associated with reproduction (Liley and Stacey, 1983; Devlin and Nagahama, 2002). Recent data from laboratory studies have shown that exposure to a variety of EDCs suppresses the sex steroids estradiol (E2) and testosterone (T) (Karels et al., 1998; Loomis and Thomas, 2000; Rocha Monteiro et al., 2000). Kime (1998) reported that the levels of sex steroids could affect the quality of eggs during gametogenesis. In this study, short-term exposure to 100 g/L butachlor caused a decrease in plasma T and E2 levels in female zebrafish. It is clear, therefore, that disturbances in sex steroids may have a considerable impact on reproductive success.
Alterations in plasma sex steroid concentrations may have resulted from several different mechanisms of action, including direct effects on steroidogenic enzymes such as aromatase, or indirect modifications associated with altered feedback loops (Mills and Chichester, 2005). Hence, the decline in T and E2 in female zebrafish might be due to inhibition of gonadal steroidogenesis by butachlor or through negative feedback on the hypothalamus–pituitary axis to reduce the synthesis of gonadotropins or to decrease cholesterol levels for steroidogenesis. In addition, sex steroids are transported to sex steroid-binding proteins in plasma resulting in a decreased rate of steroid degradation and regulation of free sex steroids available for receptor binding in different tissues (Borg, 1994). EDCs can competitively bind to steroid-binding proteins in rainbow trout (Tollefsen, 2002). The decline in T and E2 might be due to competitive binding of butachlor to steroid-binding proteins in turn increasing the clearance rate of T and E2. VTG is synthesized in the liver of female fish in response to estrogens, through binding to specific estrogen receptors (Kime, 1998), however, males do have the VTG gene, and exposure of male fish to environmental estrogens or estrogen mimics can trigger expression of the gene, leading to VTG accumulation in the blood (Ankley and Johnson, 2004; Kime, 1999). Measurement of VTG levels in males or juvenile fish is one of the most commonly used biomarkers for exposure to estrogenic chemicals in the aquatic environment (Jin et al., 2008). It was demonstrated that VTG in male zebrafish could be altered to levels above control levels in response to treatment with butachlor at concentrations as high as 100 g/L after shortterm exposure. In addition, butachlor is structurally similar to other chloracetamide herbicides (alachlor, metalochlor, and acetochlor). Some reports showed that alachlor has weak estrogenic activity (Klotz et al., 1996). Hence, our results suggested that butachlor might have estrogenic activity. The main functions of the thyroid hormones (THs) are to control growth, development, metamorphosis, reproduction, and behavior (Jugan et al., 2010; Opitz et al., 2006; Yen, 2001). Many toxic chemicals such as perfluorooctane sulfonate (PFOS), polychlorinated biphenyls (PCBs), and heavy metals, have the potential to affect thyroidal states (Bleau et al., 1996; Shi et al., 2008; Li et al., 2009). Results from mammalian toxicological studies suggest at least four classical pathways of EDC action on the thyroid system including inhibition of thyroidal iodine uptake, inhibition of thyroid peroxidase, displacement of TH from plasma transport proteins, and induction of hepatic T4 glucuronidation leading to enhanced T4 excretion (Brucker-Davis, 1998). In this work, plasma T4 and T3 levels were significantly increased in both female and male zebrafish after 30 days of exposure to butachlor. Similar results were reported for polybrominated diphenyl ethers (PBDEs) which produced a significant dose-dependent increase in circulating T3 and T4 levels in zebrafish (Kuiper et al., 2008). Exposure to HgCl2 or MeHg caused an increase in plasma T4 and T3 in juvenile rainbow trout, suggesting that Hg activates the hypothalamus–pituitary–thyroid (HPT) axis (Bleau et al., 1996). In summary, our results showed that short-term (30 days) exposure to butachlor in adult zebrafish impaired reproduction, affected the levels of thyroid hormones and sex steroids, and induced plasma VTG in males, which provides the basis for the estimated ecological risk during butachlor exposure.
Acknowledgement The authors thank Dr. Yu Cheng Zhu (USDA-ARS) for valuable comments and suggestion on the manuscript. This work was supported by the National Natural Science Foundation of China (NSFC No. 31101458).
J. Chang et al. / Experimental and Toxicologic Pathology 65 (2013) 205–209
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Effects of butachlor on reproduction and hormone levels in adult zebrafish (Danio rerio) Juhua Chang a , Shaoying Liu b , Shengli Zhou c , Minghua Wang a,∗∗ , Guonian Zhu b,∗ a Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing 210095, China b Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, China c Zhejiang Environmental Monitoring Center, Hangzhou 310015, China
a r t i c l e
i n f o
Article history: Received 3 May 2011 Accepted 18 August 2011 Keywords: Butachlor Reproduction Sex steroid Thyroid hormone Vitellogenin Zebrafish
a b s t r a c t Butachlor, a chloracetamide herbicide, is widely used in China. In the present study, paired adult male and female zebrafish (Danio rerio) were exposed to various concentrations of butachlor (0, 25, 50 and 100 g/L) for 30 days, and the effects on reproduction and endocrine disruption were evaluated using fecundity, condition factor (CF), gonadosomatic index (GSI), liver somatic index (LSI), plasma vitellogenin (VTG), sex steroids and thyroid hormone levels as endpoints. Our results showed that the mean fecundity rates were significantly decreased at 50 and 100 g/L butachlor during the 30-day exposure period. At the end of the exposure period, no significant changes were observed in CF and LSI in both females and males, while GSI was significantly reduced in males at 50 and 100 g/L butachlor. At 100 g/L butachlor, plasma testosterone (T) and 17-estradiol (E2) levels were significantly decreased in females, while plasma VTG level was significantly increased in males. Plasma thyroxine (T4) and triiodothyronine (T3) levels were significantly increased at 50 and 100 g/L butachlor in males, and at 100 g/L in females. This work demonstrated that butachlor adversely affected the normal reproductive success of zebrafish, and disrupted the thyroid and sex steroid endocrine systems, which provides the basis for the estimated ecological risk during butachlor exposure. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Man-made pesticides are among the most common sources of environmental pollutants worldwide (Williams, 1996). Butachlor is a chloracetamide herbicide, and is one of the top three herbicides extensively applied to control weeds in rice fields in China (Yu et al., 2003). The herbicide has contaminated river water via the effluents from rice paddy fields (Ohyama et al., 1987). Residues of this herbicide are present in groundwater, including that used in some cases for human consumption (Natarajan, 1993). Butachlor is persistent in agricultural soil and water systems (Yu et al., 2003), posing a potential threat to the agro-ecosystem and human health (Debnath et al., 2002; Sinha et al., 1995). Most experiments investigating the effects of butachlor have focused on genotoxicity to fish, amphibians and rats (Ateeq et al., 2002, 2006; Geng et al., 2005), and some investigations have indicated that butachlor is a suspected carcinogen (Ou et al., 2000). Tomlin reported on the acute toxicity of butachlor to fish, with LC50 (96 h) values ranging from 0.14 to
∗ Corresponding author. Tel.: +86 0571 86971220; fax: +86 0571 86971902. ∗∗ Corresponding author. Tel.: +86 025 84395479; fax: +86 025 84395479. E-mail addresses: [email protected] (M. Wang), [email protected] (G. Zhu). 0940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2011.08.007
0.52 mg/L, which showed that butachlor was highly toxic to fish (Tomlin, 1994). However, little is known about the reproductive toxicity of butachlor. A short-term fish reproduction assay has been developed for detecting the effects of endocrine-disrupting compounds (EDCs) (Ankley et al., 2001, 2002). A suite of biomarkers is needed to best assess possible disturbances of the endocrine system in wildlife. The use of vitellogenin (VTG) to screen for estrogen exposure in oviparous vertebrates is well-described (Denslow et al., 1999; Harries et al., 2000; Jobling et al., 2003; Korte et al., 2000). Plasma sex steroids (T and E2) have also been used in the study of EDCs in fish (Ankley et al., 2001, 2002). In fish, growth and reproduction are believed to be, at least partly, under the control of the thyroid hormones, thyroxine (T4) and triiodothyronine (T3) (Cyr and Eales, 1996; Power et al., 2001). The zebrafish (Danio rerio) is an attractive model organism for evaluating reproductive toxicity and endocrine-disrupting effects because of its small size, ease of culture, short life cycle and prolific egg production with high fertilization and hatching rates (Segner, 2009). This study was conducted to evaluate the effects of butachlor on reproduction and endocrine disruption in adult zebrafish using a short-term (30 days) assay. In this assay, fecundity was assessed during the exposure period. At the end of the exposure period, this
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study integrated different endpoints such as plasma vitellogenin (VTG), sex steroids (T and E2), and thyroid hormones (T4 and T3) levels, gonadosomatic index (GSI), liver somatic index (LSI) and condition factor (CF) of the zebrafish.
Table 1 Condition factor (CF), gonadosomatic index (GSI) and liver somatic index (LSI) for adult male and female zebrafish after 30 days of exposure to butachlor (n = 15). Index
Sex
Control
CF (%)
♀ ♂ ♀ ♂ ♀ ♂
1.47 1.26 13.10 1.26 2.81 1.59
2. Materials and methods GSI (%)
2.1. Chemicals Butachlor (technical grade AI: 96%) was purchased from Hangzhou Qingfeng Agrochemical Co., Ltd. Butachlor was dissolved in dimethyl sulfoxide (DMSO), and the stock solution (100,000 mg/L) was stored in the dark at 4 ◦ C. MS-222 (3aminobenzoic acid ethyl ester, methanesulfonate salt) was purchased from Sigma (St. Louis, MO, USA). All other chemicals used were analytical grade. 2.2. Test fish and culture conditions Adult zebrafish (D. rerio) were purchased from a local fish shop. The fish were fed with freshly hatched brine shrimp twice a day and were maintained at 28 ± 1 ◦ C and a 14:10 h light:dark photoperiod. Prior to exposure to the herbicide, 100 pairs of zebrafish (one pair/tank) were set up to assess reproductive viability over three weeks, and egg production was recorded daily. Sixty pairs of zebrafish were then selected for butachlor exposure according to fecundity during the three weeks of acclimation. 2.3. Exposure and experimental design Adult zebrafish were exposed to various concentrations (0, 25, 50 and 100 g/L) of butachlor for 30 days, and fifteen pairs of fish were included in the control group and the butachlor-treatment group. One male and one female were set up in a 2 L spawning tank, containing 1.5 L test solution, which was changed daily. Both the exposure and control group received 0.001% (v/v) DMSO. Eight pairs in each treatment were selected to evaluate the effects of butachlor on the fecundity of zebrafish. Two hour after the light had gone on in the morning, spawned eggs were collected and counted from each tank daily. Fecundity rate (as the mean number of eggs per female per day), oviposition (spawning) frequency per female, the mean number of eggs per female per spawning and cumulatively per dose were calculated for the whole reproductive test period (30 days) for each treatment. After 30 days of exposure, all fish were anesthetized with 0.03% MS-222. After measurement of body weight and total length, the gonads and livers were removed and weighed. The gonadosomatic index (GSI = 100·[gonad weight (g)/body weight (g)]), liver somatic index (LSI = 100·[liver weight (g)/body weight (g)]), and condition factor (=100·[body weight (g)/total length3 (cm)]) were calculated. Blood samples were collected from the caudal vein of each fish into heparinized microcapillary tubes, and the blood samples from five fish of the same sex were pooled as one replicate. Three replicates for the control and exposure groups were obtained. After volume measurement, a 5-fold volume of phosphate buffer (10 mM K2 HPO4 , 100 mM KCl, 1 mM EDTA, 1 Mm DTT, pH 7.4) was added to each tube. The samples were immediately centrifuged (10,000 × g, 10 min, 4◦ C), and the obtained plasma samples were stored at −80 ◦ C until analysis.
LSI (%) *
± ± ± ± ± ±
25 g/L 0.17 0.04 1.13 0.19 0.58 0.41
1.57 1.47 10.97 0.85 2.54 1.15
± ± ± ± ± ±
50 g/L 0.07 0.09 0.70 0.20 0.21 0.13
1.73 1.40 10.13 0.80 2.07 1.51
± ± ± ± ± ±
100 g/L 0.11 0.05 0.88 0.08* 0.22 0.24
1.57 1.38 11.39 0.68 1.89 1.40
± ± ± ± ± ±
0.15 0.09 1.31 0.11* 0.24 0.37
Significant difference compared with the control (P < 0.05).
2.5. Statistical analysis Data were statistically analyzed using the SAS program (SAS, 1990) and the Proc GLM procedure was used for variance analyses. Mean separation was conducted using SAS ProcMeans/LSD procedures. The value P < 0.05 was used as the criterion for statistical significance. All data are expressed as mean ± SE. 3. Results 3.1. Effects of butachlor on condition factor, gonadosomatic index and liver somatic index in zebrafish No mortality was observed in any of the treatment groups during the exposure period. Butachlor did not affect condition factor (CF) or liver somatic index (LSI) in females or males in the butachlor-treated groups after 30 days of exposure (Table 1). Reduced gonadosomatic index (GSI) was observed in males in the 50 and 100 g/L butachlor-treated groups compared to the control fish (F = 3.53, P = 0.0236; Table 1). In female fish, no significant differences in the GSI between the control and treatment groups were observed (F = 1.51, P > 0.05; Table 1). 3.2. Effects of butachlor on reproduction in zebrafish Spawning events and egg production were assessed during the 30-day exposure period. Total egg production ranged from 2225 to 7118 across all treatments. Each female produced 278–890 eggs. The mean fecundity rates (eggs/female/day) in the 50 and 100 g/L treatments were 15.46 ± 3.42 and 7.32 ± 2.35, respectively, which were significantly lower than that of the control (27.49 ± 5.89; F = 6.02, P = 0.0027; Fig. 2A). The significant reduction in cumulative eggs produced in the 50 and 100 g/L treatments (Fig. 1) was apparently associated with the reduced number of eggs per spawn
2.4. Plasma hormones and vitellogenin measurement Plasma T, E2, VTG, T4 and T3 levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (RD Chemical, Mountain View, CA, USA), following the manufacturer’s instructions.
Fig. 1. Cumulative number of eggs produced by zebrafish breeding pairs exposed to various concentrations of butachlor for 30 days (n = 8).
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Fig. 2. Reproductive parameters of zebrafish breeding pairs exposed to various concentrations of butachlor for 30 days. (A) Eggs per female per day, (B) eggs per female per spawning, and (C) the number of spawning event per female. Data are expressed as the mean ± SE. (n = 8), *significant difference compared with the control (P < 0.05).
(F = 21.06, P < 0.0001; Fig. 2B) and the reduced number of spawning events (F = 6.23, P = 0.0022; Fig. 2C).
3.3. Effects of butachlor on plasma sex steroid and vitellogenin levels in zebrafish The results of the zebrafish plasma sex steroid and vitellogenin analyses are summarized in Fig. 3. After 30 days of exposure, plasma T and E2 levels were significantly decreased in females at 100 g/L butachlor (for T, F = 5.24, P = 0.0271; for E2, F = 2.52, P = 0.1319; Fig. 3A and B), while no significant differences in plasma T and E2 levels in males were observed between the exposure and control groups (for T, F = 0.63, P > 0.05; for E2, F = 1.14, P > 0.05; Fig. 3A and B). Moreover, exposure to 100 g/L butachlor induced a significantly elevated level of VTG in males (F = 13.51, P = 0.0011; Fig. 3C), but did not affect VTG level in females (F = 0.77, P > 0.05; Fig. 3C).
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Fig. 3. Plasma levels of (A) T, (B) E2 and (C) VTG in zebrafish after 30 days of exposure to butachlor. Data are expressed as the mean ± SE. (five fish as a pool, n = 3), *significant difference compared with the control (P < 0.05).
3.4. Effects of butachlor on plasma thyroid hormone levels in zebrafish The results of the zebrafish plasma thyroid hormone analyses are summarized in Fig. 4. At the end of the exposure period, plasma T4 and T3 concentrations in males were significantly increased in the 50 and 100 g/L butachlor-treated groups compared to the control group (for T4, F = 3.26, P = 0.0807; for T3 F = 44.51, P < 0.0001; Fig. 4A and B); In females, plasma T3 and T4 concentrations were significantly increased in the 100 g/L butachlor-treated group compared with the control group (for T4, F = 6.01, P = 0.019; for T3, F = 49.38, P < 0.0001; Fig. 4A and B). 4. Discussion Environmental chemicals that interfere with reproduction in fish have attracted significant attention in recent studies (Ankley et al., 2001, 2002). In the present study, a short-term (30 days) reproduction assay were performed to evaluate the effects of
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Fig. 4. Plasma levels of (A) T4 and (B) T3 in zebrafish after 30 days of exposure to butachlor. Data are expressed as the mean ± SE. (five fish as a pool, n = 3), *significant difference compared with the control (P < 0.05).
butachlor on reproduction and endocrine disruption in adult zebrafish, and ELISA was used to analyze the changes in plasma T, E2, VTG, T4 and T3 levels. It has been proposed that condition factor (CF) can be used as a non-specific measure of physiological fitness in fish health (Anderson et al., 2003), because CF is reflective of changes in food intake, fat deposition, and protein budgets (Smolders et al., 2003). In our study, butachlor did not affect CF in both females and males in all butachlor-treated groups after 30 days exposure. Therefore, the effects of butachlor on reproductive output were clearly sublethal and might be mediated via endocrine disruption. Reproduction is dependent upon the appropriate coordination of the entire hypothalamus–pituitary–gonad (HPG) axis, therefore, chemicals acting at any level of this axis via any mechanism can have adverse effects on reproduction (Brian et al., 2007). Our results indicated that the fecundity rates in the 50 and 100 g/L butachlor-treatments were significantly decreased during the 30-day exposure period. Sex steroids play a crucial role in sex differentiation, sexual maturation, and various female behaviors associated with reproduction (Liley and Stacey, 1983; Devlin and Nagahama, 2002). Recent data from laboratory studies have shown that exposure to a variety of EDCs suppresses the sex steroids estradiol (E2) and testosterone (T) (Karels et al., 1998; Loomis and Thomas, 2000; Rocha Monteiro et al., 2000). Kime (1998) reported that the levels of sex steroids could affect the quality of eggs during gametogenesis. In this study, short-term exposure to 100 g/L butachlor caused a decrease in plasma T and E2 levels in female zebrafish. It is clear, therefore, that disturbances in sex steroids may have a considerable impact on reproductive success.
Alterations in plasma sex steroid concentrations may have resulted from several different mechanisms of action, including direct effects on steroidogenic enzymes such as aromatase, or indirect modifications associated with altered feedback loops (Mills and Chichester, 2005). Hence, the decline in T and E2 in female zebrafish might be due to inhibition of gonadal steroidogenesis by butachlor or through negative feedback on the hypothalamus–pituitary axis to reduce the synthesis of gonadotropins or to decrease cholesterol levels for steroidogenesis. In addition, sex steroids are transported to sex steroid-binding proteins in plasma resulting in a decreased rate of steroid degradation and regulation of free sex steroids available for receptor binding in different tissues (Borg, 1994). EDCs can competitively bind to steroid-binding proteins in rainbow trout (Tollefsen, 2002). The decline in T and E2 might be due to competitive binding of butachlor to steroid-binding proteins in turn increasing the clearance rate of T and E2. VTG is synthesized in the liver of female fish in response to estrogens, through binding to specific estrogen receptors (Kime, 1998), however, males do have the VTG gene, and exposure of male fish to environmental estrogens or estrogen mimics can trigger expression of the gene, leading to VTG accumulation in the blood (Ankley and Johnson, 2004; Kime, 1999). Measurement of VTG levels in males or juvenile fish is one of the most commonly used biomarkers for exposure to estrogenic chemicals in the aquatic environment (Jin et al., 2008). It was demonstrated that VTG in male zebrafish could be altered to levels above control levels in response to treatment with butachlor at concentrations as high as 100 g/L after shortterm exposure. In addition, butachlor is structurally similar to other chloracetamide herbicides (alachlor, metalochlor, and acetochlor). Some reports showed that alachlor has weak estrogenic activity (Klotz et al., 1996). Hence, our results suggested that butachlor might have estrogenic activity. The main functions of the thyroid hormones (THs) are to control growth, development, metamorphosis, reproduction, and behavior (Jugan et al., 2010; Opitz et al., 2006; Yen, 2001). Many toxic chemicals such as perfluorooctane sulfonate (PFOS), polychlorinated biphenyls (PCBs), and heavy metals, have the potential to affect thyroidal states (Bleau et al., 1996; Shi et al., 2008; Li et al., 2009). Results from mammalian toxicological studies suggest at least four classical pathways of EDC action on the thyroid system including inhibition of thyroidal iodine uptake, inhibition of thyroid peroxidase, displacement of TH from plasma transport proteins, and induction of hepatic T4 glucuronidation leading to enhanced T4 excretion (Brucker-Davis, 1998). In this work, plasma T4 and T3 levels were significantly increased in both female and male zebrafish after 30 days of exposure to butachlor. Similar results were reported for polybrominated diphenyl ethers (PBDEs) which produced a significant dose-dependent increase in circulating T3 and T4 levels in zebrafish (Kuiper et al., 2008). Exposure to HgCl2 or MeHg caused an increase in plasma T4 and T3 in juvenile rainbow trout, suggesting that Hg activates the hypothalamus–pituitary–thyroid (HPT) axis (Bleau et al., 1996). In summary, our results showed that short-term (30 days) exposure to butachlor in adult zebrafish impaired reproduction, affected the levels of thyroid hormones and sex steroids, and induced plasma VTG in males, which provides the basis for the estimated ecological risk during butachlor exposure.
Acknowledgement The authors thank Dr. Yu Cheng Zhu (USDA-ARS) for valuable comments and suggestion on the manuscript. This work was supported by the National Natural Science Foundation of China (NSFC No. 31101458).
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