Endocrine Disrupting Compounds
Subject Area 6.4
Area 6.4 • Monitoring and Fate of Persistent Chemicals Research Article
Selected Endocrine Disrupting Compounds (Vinclozolin, Flutamide, Ketoconazole and Dicofol): Effects on Survival, Occurrence of Males, Growth, Molting and Reproduction of Daphnia magna Maher H. Haeba1, Klára Hilscherová1,2 , Edita Mazurová1 and Ludek Bláha1,2* 1 RECETOX
– Research Centre for Environmental Chemistry and Ecotoxicology, Masaryk University, Kamenice 3, 62500 Brno, Czech Republic 2 Institute of Botany, Czech Academy of Sciences, Kvetná 8, 60325 Brno, Czech Republic * Corresponding author (
[email protected]) DOI: http://dx.doi.org/10.1065/espr2007.12.466 Please cite this paper as: Haeba MH, Hilscherová K, Mazurová E, Bláha L (2008): Selected Endocrine Disrupting Compounds (Vinclozolin, Flutamide, Ketoconazole and Dicofol): Effects on Survival, Occurrence of Males, Growth, Molting and Reproduction of Daphnia magna. Env Sci Pollut Res 15 (3) 222–227 Abstract
Background, Aim and Scope. Pollution–induced endocrine disruption in vertebrates and invertebrates is a worldwide environmental problem, but relatively little is known about effects of endocrine disrupting compounds (EDCs) in planktonic crustaceans (including Daphnia magna). Aims of the present study were to investigate acute 48 h toxicity and sub-chronic (4–6 days) and chronic (21 days) effects of selected EDCs in D. magna. We have investigated both traditional endpoints as well as other parameters such as sex determination, maturation, molting or embryogenesis in order to evaluate the sensitivity and possible use of these endpoints in ecological risk assessment. Materials and Methods. We have studied effects of four model EDCs (vinclozolin, flutamide, ketoconazole and dicofol) on D. magna using (i) an acute 48 h immobilization assay, (ii) a sub-chronic, 4–6 day assay evaluating development and the sex ratio of neonates, and (iii) a chronic, 21 day assay studying number of neonates, sex of neonates, molting frequency, day of maturation and the growth of maternal organisms. Results. Acute EC50 values in the 48 h immobilization test were as follows (mg/L): dicofol 0.2, ketoconazole 1.5, flutamide 2.7, vinclozolin >3. Short-term, 4–6 day assays with sublethal concentrations showed that the sex ratio in Daphnia was modulated by vinclozolin (decreased number of neonate males at 1 mg/L) and dicofol (increase in males at 0.1 mg/L). Flutamide (up to 1 mg/L) had no effect on the sex of neonates, but inhibited embryonic development at certain stages during chronic assay, resulting in abortions. Ketoconazole had no significant effects on the studied processes up to 1 mg/L. Discussion. Sex ratio modulations by some chemicals (vinclozolin and dicofol) corresponded to the known action of these compounds in vertebrates (i.e. anti-androgenicity and anti-oestrogenicity, respectively). Our study revealed that some chemicals known to affect steroid-regulated processes in vertebrates can
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also affect sublethal endpoints (e.g. embryonic sex determination and/or reproduction) in invertebrates such as D. magna. Conclusions. A series of model vertebrate endocrine disrupters affected various sub-chronic and chronic parameters in D. magna including several endpoints that have not been previously studied in detail (such as sex determination in neonates, embryogenesis, molting and maturation). Evaluations of traditional reproduction parameters (obtained from the 21 day chronic assay) as well as the results from a rapid, 4–6 day, sub-chronic assay provide complementary information on non-lethal effects of suspected organic endocrine disrupters. Recommendations and Perspectives. It seems that there are analogies between vertebrates and invertebrates in toxicity mechanisms and in vivo effects of endocrine disruptors. However, general physiological status of organisms may also indirectly affect endpoints that are traditionally considered 'hormone regulated' (especially at higher effective concentrations as observed in this study) and these factors should be carefully considered. Further research of D. magna physiology and comparative studies with various EDCs will help to understand mechanisms of action as well as ecological risks of EDCs in the environment. Keywords: Daphnia magna; dicofol; endocrine disruption;
flutamide; ketoconazole; sex determination; vinclozolin
Introduction
Anthropogenic chemicals have been shown to cause endocrine disruption in numerous organisms. Endocrine disruptive chemicals (EDCs) have caused a wide range of effects in wildlife and possibly in humans (Tyler et al. 1998, StahlschmidtAllner et al. 1997, Basler & Lebsanft 1999, Keiter et al. 2006). There are also numerous EDC-induced effects documented in invertebrates, including planktonic crustaceans (Hense et al. 2005, LeBlanc 2007). The most often reported effects include an alteration in testosterone metabolism (Baldwin & Leblanc 1994, Baldwin et al. 1998), which could lead to imposex or intersex development, perturbations in the molt cycle (Zou & Fingerman 1997), growth retardation (LeBlanc & McLachlan 1999), developmental abnormalities (Olmstead & LeBlanc 2000) or modulations of fecundity (Bryan et al. 1986). In crustaceans, some ef-
Env Sci Pollut Res 15 (3) 222 – 227 (2008) © Springer 2008
Subject Area 6.4 fects of EDCs seem to be mediated by steroid or ecdysteroid regulated processes acting via intercellular receptors and transcription factors in a way similar to vertebrates (Chang 1993, LeBlanc & McLachlan 1999, Subramonian 2000). Daphnia magna is one of the most often used organisms in ecotoxicology, and it has been evaluated as a model for studies of endocrine disruption (Kashian & Dodson 2004, Sanchez et al. 2005). Fecundity, growth rate and maturation are some of the parameters that might be affected by EDCs. Also the sex ratio (proportion of males in the population) has been shown to be a sensitive indicator of stress factors, including chemical pollutants (Dodson et al. 1999b). Daphnia magna reproduce mostly by parthenogenesis and females are usually dominant in daphnid populations. It has been found that juvenile hormone III and methyl farnesoate as well as their chemical analogs used as pesticides (such as pyriproxyfen and fenoxycarb) increase occurrence of male daphnids in the population (Olmstead and Leblanc 2002, Olmstead and LeBlanc 2003, Tatarazako et al. 2003, Wang et al. 2005). Similarly, a recent study reported that two insect juvenile hormones (JH I and JH II) and three juvenile hormone analogs (kinoprene, hydroprene and epofenonane) increased the proportion of males in the Daphnia magna population (Oda et al. 2005). The increase in the sex ratio has also been observed in Daphnia exposed to such chemicals as atrazine or acetone (Dodson et al. 1999). On the other hand, other compounds (e.g. methoprene and dieldrin) may decrease male production in crustaceans by mimicking or interfering with methyl farnesoate action (Peterson et al. 2001, Dodson et al. 1999). Despite some previous studies, our understanding on the sublethal effects of possible EDCs in invertebrates is still limited. In the present study we have investigated effects of four chemicals that are suspected of interfering with normal reproduction and development in Daphnia; vinclozolin (dicarboximide fungicide known to act as an antagonist of androgen receptors in vertebrates; Sperry & Thomas 1999), flutamide (a drug clinically used in treatment of human prostate cancer acting as an anti-androgen; Kolvenbag et al. 2001), dicofol (an organochlorine acaricide manufactured from technical DDT known to be antioestrogenic in vertebrates; Vinggaard et al. 2000), and ketoconazole (an anti-fungal imidazole derivative that inhibits various CYP enzymes, acting also as an anti-androgen; Gray et al. 1999). The major goals of our study were to explore both acute toxicity and effects of sublethal doses in the sub-chronic (4–6 days) and chronic (21 days) assays with D. magna. We focused on several traditional endpoints as well as on less frequently employed parameters such as sex ratio, maturation, molting or embryogenesis, in order to evaluate the sensitivity of these parameters and their possible use in the ecological risk assessment. 1 1.1
Materials and Methods Material
Daphnia magna (long-term laboratory culture originally collected from a freshwater reservoir in Brno, Czech Republic) have been permanently maintained for more than 3 years under controlled conditions: temperature 20 ± 2ºC, 16/8 hr light/
Env Sci Pollut Res 15 (3) 2008
Endocrine Disrupting Compounds dark cycle in the Elendt M4 medium (Samel et al. 1999, OECD 1996). Dimethylsulfoxide (DMSO) of analytical grade (99% purity) was used as a non-toxic solvent at 0.05% v/v. All tested chemicals (vinclozolin, flutamide, ketoconazole and dicofol) were purchased from Sigma-Aldrich. 1.2
Acute toxicity testing
In acute toxicity tests, neonates less than 24 h old (twenty animals for each treatment and control) were used. The exposure medium (8.88 g CaCl2, 2.4 g MgSO4, 2.59 g NaHCO3 and 0.23 g KCl per litre of water) was not renewed during the test and organisms were not fed. Mortality (immobilization) was recorded after 24 and 48 hours. 1.3
Sub-chronic toxicity testing
Sub-chronic toxicity test was conducted with gravid (10– 14-day-old adults) females with the first eggs in their brood chamber. Daphnids were examined microscopically for developmental stage of embryos and females having late embryonic maturation stages (Kast-Hutcheson et al. 2001) were used for experiments (exposed to sublethal doses estimated from the acute toxicity assays). The first batch of neonates (hatching within the first 24 h) was always discarded as these animals were not exposed to the tested chemicals during their entire developmental period. Neonates (from the second brood) spent their entire embryonic development under exposure to tested chemicals and they were used for toxicity evaluation. Ten replicate polypropylene jars (each with individual D. magna females in 50ml medium covered with saran wrap to prevent volatilization) were used per treatment. Exposure medium was renewed every 48h. The offspring was removed daily and counted. Development and the sex of neonates were assessed using a low magnification light microscope. Offspring males were identified by the presence of prominent first antennules and the sex ratio was calculated as the number of males divided by the total number of neonates (Dodson et al. 1999). Assay was terminated after all females released the second brood of neonates (typically 4–6 days of exposure). 1.4
Chronic reproduction assay
Chronic, 21 day, toxicity assays were started with neonate females younger than 24 h placed individually into beakers with 50 ml of M4 medium. Ten replicate jars (each with an individual D. magna female; covered with saran wrap to prevent volatilization) were used per treatment and the exposure medium was renewed every 48 h. The following parameters were evaluated: offspring counts and their sex, molting frequency, day of maturation (i.e. time to the first reproduction) and the lengths of maternal organisms (on days 0, 7, 15 and 21; LeBlanc & McLachlan 1999). The daphnids in the sub-chronic and chronic assays were fed every other day (at the time of medium exchange) with a Selenastrum capricornutum and Chlorella kessleri mixture (107 cells in 1 ml administered into 50 ml jar). Feeding habits during experiments were monitored and no differences between controls and exposed animals were recorded.
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Endocrine Disrupting Compounds
1.5
Subject Area 6.4
Stability of tested compounds Ketokonazole
Dicofol
Immobilization (% control)
Stability of the tested compounds during 48 h (renewal period of the exposure media) was checked by monitoring changes in UV-VIS spectra (scan of 200–600 nm with Varian CARY cuvette spectrophotometer). For flutamide and vinclozolin, the results were confirmed by Ultra Performance Liqmilli-Q water was used. The compounds were detected by absorbance monitoring at a range of 200–300 nm. Analyses were performed using validated methods by the contract partner (pharmaceutical company Pliva-Lachema, a.s., Brno, Czech Republic). Less than 15% decrease in concentrations of all tested compounds was observed during a 48 h period of media exchange and nominal concentrations were used for calculations of toxicity values.
Flutamide
100
75
50
25
0 -1
0
concentration (log mg/L)
1.6
Statistics
Fig. 1: Acute toxicity of the tested compounds to D. magna (48 h immobilization, concentration-response curves). Vinclozolin was not toxic up to its water solubility (3 mg/L)
The 48 h EC50 values were calculated by Probit analysis (Finney 1971). Statistical comparisons between exposure groups were performed by one way analysis of variance (ANOVA) followed by Dunnet's test to detect differences among treatment groups in comparison with controls. Nonparametric tests were employed when the data were heterogeneous. Statistics were calculated in Statistica for Windows 6.0. P-values less than 0.05 were considered statistically significant. 2
Table 1: Acute effects (24 and 48 h EC50 values) of the tested compounds on immobilization in D. magna (EC50 in mg/L; 95% confidence limits for EC50 (in parentheses)) Acute toxicity to D. magna (EC50; mg/L) 24 h
48 h
Dicofol
0.38 (0.32–0.46)
0.2 (0.17–0.24)
Ketoconazole
8.1 (4.6–10.8)
1.51 (1.16–1.91)
Flutamide
7.8 (5.9–28.4)
2.7 (2.15–3.41)
Vinclozolin
>3
>3
Results
Full dose-response curves for acute toxicity are in Fig. 1 and estimated EC50 values for tested compounds are presented in Table 1. Dicofol had the highest toxicity with 48 h EC50 of 0.2 mg/L, while vinclozolin was the least toxic with no effect on immobilization up to its solubility (> 3 mg/L). Concentrations causing no significant effects (No Observed Effects Concentrations – NOECs) in the acute tests were selected for further sub-chronic and chronic assays, whose results are summarized in Table 2. No mortalities were observed during sub-chronic and chronic exposures. Ketoconazole (up to 1.0 mg/L) had no significant effect on any of the investigated parameters.
creased the sex ratio in favour of males at the highest concentration tested, 0.1 mg/L (Fig. 2A), while vinclozolin (1 mg/L) decreased the number of neonate males (Fig. 2B). Chronic, 21 day exposures to the highest concentration of flutamide (1.0 mg/L) significantly (p
