Aquatic Toxicology 157 (2014) 141–149
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Transient exposure to environmental estrogen affects embryonic development of brown trout (Salmo trutta fario) Sara Schubert a,b,c,∗ , Armin Peter a , René Schönenberger d , Marc J.-F. Suter d , Helmut Segner e , Patricia Burkhardt-Holm b,f a Eawag – Swiss Federal Institute of Aquatic Science and Technology, Department of Fish Ecology and Evolution, Seestrasse 79, CH-6047 Kastanienbaum, Switzerland b Man–Society–Environment, Department of Environmental Sciences, University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland c Institute of Clinical Pharmacology, Medical Faculty Carl Gustav Carus, Technical University of Dresden, Germany d Eawag – Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Toxicology, Ueberlandstrasse 133, CH-8600 Duebendorf, Switzerland e Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, CH-3001 Bern, Switzerland f Department of Biological Sciences, University of Alberta, Edmonton, Canada
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Article history: Received 20 June 2014 Received in revised form 1 October 2014 Accepted 12 October 2014 Available online 22 October 2014 Keywords: Fish early life stage Developmental toxicity 17-estradiol Hatching Heart rate Body length
a b s t r a c t Transient exposure of brown trout embryos from fertilization until hatch (70 days) to 17-estradiol (E2) was investigated. Embryos were exposed to 3.8 and 38.0 ng/L E2 for 2 h, respectively, under four scenarios: (A) exposure once at the day of fertilization (0 days post-fertilization, dpf), (B) once at eyeing stage (38 dpf), (C) weekly exposure until hatch or (D) bi-weekly exposure until hatch. Endpoints to assess estrogen impact on embryo development were fertilization success, chronological sequence of developmental events, hatching process, larval malformations, heart rate, body length and mortality. Concentration-dependent acceleration of development until median hatch was observed in all exposure scenarios with the strongest effect observed for embryos exposed once at 0 dpf. In addition, the hatching period was significantly prolonged by 4–5 days in groups receiving single estrogen exposures (scenarios A and B). Heart rate on hatching day was significantly depressed with increasing E2 concentrations, with the strongest effect observed for embryos exposed at eyeing stage. Estrogenic exposure at 0 dpf significantly reduced body length at hatch, not depending on whether this was a single exposure or the first of a series (scenarios A and D). The key finding is that even a single, transient E2 exposure during embryogenesis had significant effects on brown trout development. Median hatch, hatching period, heart rate and body length at hatch were found to be highly sensitive biomarkers responsive to estrogenic exposure during embryogenesis. Treatment effects were observable only at the post-hatch stage. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The presence of endocrine disrupting compounds (EDCs) in the aquatic environment is well documented worldwide (Luo et al., 2014; Sun et al., 2014). Both, exposure to and effects of
∗ Corresponding author at: Institute of Clinical Pharmacology, Technical University of Dresden, Fiedlerstrasse 27, 01307 Dresden, Germany. Tel.: +49 351 4584534; fax: +49 351 458 4341. E-mail addresses:
[email protected] (S. Schubert),
[email protected] (A. Peter),
[email protected] (R. Schönenberger),
[email protected] (M.J.-F. Suter),
[email protected] (H. Segner),
[email protected] (P. Burkhardt-Holm). http://dx.doi.org/10.1016/j.aquatox.2014.10.007 0166-445X/© 2014 Elsevier B.V. All rights reserved.
estrogen-active EDCs (natural and synthetic estrogens as well as xenoestrogens) have repeatedly been reported for fish (Jobling and Tyler, 2003; Sumpter and Johnson, 2008; Schultz et al., 2013). The impacts of EDCs on fish have been studied primarily with respect to their effects on sexual development and adult reproduction. However, exposure to EDCs can occur during all life stages of fish, including early life stages before sexual differentiation. Early life stages in fish are considered to be most sensitive against pollutants (von Westernhagen, 1988; Heath, 1995; Babin et al., 2007), although fish embryos may not generally be more sensitive than adult fish (Belanger et al., 2013). Accordingly, the sensitivity of early fish life stages has to be determined. The question is then whether EDC exposure would still exert toxic effects in early life stages, for instance impairing survival and growth. In Chinese rare
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minnow (Gobiocypris rarus) fry mortality at the swim-up stage was affected by chronic exposure to 16 ng/L 17␣-ethinylestradiol (EE2; initiated at 5 mm in brown trout) and long developmental periods, characteristic for salmonids, may favour increased uptake of toxicants during the incubation period in the gravel bed (Kristensen, 1994; Kime, 1998). Besides effects of (xeno-)estrogens on sexual differentiation and reproduction in trout (Körner et al., 2008; Schubert et al., 2008; Brown et al., 2009), estrogenic exposure may act as a stressor for other developmental processes such as hatching and lead to developmental abnormalities as described for other species and substances (Jin et al., 2009). In field studies, polluted river water with estrogenic potency (Körner et al., 2001) led to lower heart rate as well as retarded development and reduced growth rate in embryonic stages of brown trout (Luckenbach et al., 2001a,b). Which specific pollutants caused these adverse effects in the mentioned studies, however, could not be determined. To unravel environmental complexity and to identify cause–effect relationships, field studies need to be completed with laboratory experiments using defined exposure scenarios. Most often such studies are performed in a continuous exposure regime, while the actual situation in the environment is subject to dynamic variations (Vermeirssen et al., 2006; Martinovic´ et al., 2008; Nelson et al., 2011). One rare exception taking into account the dynamic behaviour of the system is a study by Knudsen and colleagues where E2 uptake and vitellogenin induction was monitored in juvenile brown trout exposed to 3–6 h pulses of E2 at concentrations of up to 370 ng/L (Knudsen et al., 2011). To date no investigations on effects of pulsed estrogen exposure on embryonic development of brown trout have been performed at environmentally relevant concentrations. Furthermore, it is not yet clear which time window during embryonic development is especially sensitive for survival in this cold water species with its long temperature-depended embryogenesis. This is especially important because salmonid fish species have been found to be declining in the wild (Burkhardt-Holm et al., 2005; Keiter et al., 2006). The aim of the study was to (i) elucidate whether 17-estradiol (E2) has toxic effects on brown trout embryos when exposed to environmentally relevant concentrations showing a dynamic behaviour as often observed in the environment (Vermeirssen et al., 2006; Martinovic´ et al., 2008; Nelson et al., 2011), (ii) identify the most sensitive time points for estrogenic exposure and (iii) find which of the examined endpoints are the most sensitive to estrogen exposure in brown trout under the given scenario. The following endpoints were assessed during embryogenesis and in freshly hatched brown trout: fertilization success, mortality, malformations, growth of eggs, hatching process (median hatch, time period of hatching), heart rate and body length of eleutheroembryos. Consequently, the present study focused on non-endocrine, toxicological endpoints since ecological recruitment success during the stages from freshly fertilized until freshly hatched brown trout is driven by parameters like hatching rate and survival and endocrine parameters may become relevant later in life.
2. Materials and methods 2.1. Brown trout embryos Mature 3-year-old female and male brown trout (Salmo trutta fario) were obtained from a private hatchery (Mändli, Liestal, Switzerland). Fish were held overnight in the laboratory facilities at Lake Lucerne. For the fertilization procedure, eggs and sperm were stripped the next day (February 1st) from slightly narcotized fish (narcotizing mixture: 30 L lake water with 0.5 mL clove oil dissolved in 10 mL ethanol), mixed and incubated in lake water for 30 min until hardening was completed (Schubert et al., 2008). Eggs from five females (TL 352.6 ± 18.3 mm) and sperm from four males (TL 351.0 ± 5.1 mm) were taken for exposure scenarios A and B (see Section 2.2) and eggs from six females (TL 356.0 ± 7.8 mm) and sperm from four males (TL 329.3 ± 19.6 mm) were taken for exposure scenarios C and D (see Section 2.2). Different parent pools were used to simplify the exposure handling on fertilization day. Directly after hardening was completed, 250 fertilized eggs were randomly distributed into each sieve (stainless steel, mesh size of 1.56 mm, Peternier AG, Kriens, Switzerland) to enable separate handling and to minimize mechanical disturbance during the exposure. Developing eggs remained in the sieves during the entire experiment (until hatch). Sieves loaded with eggs were transferred into a vertical flow egg incubation system (a brood device normally used in fish hatcheries to breed salmonid eggs; Vetterli Brutschränke, Switzerland) in complete darkness with a flow rate of 960–1120 mL/min lake water and an average water temperature of 6.1 ◦ C (min 5.8 ◦ C, max 6.5 ◦ C) during the incubation time. For the estrogenic exposure, egg sieves were temporarily transferred to exposure aquaria. Lake water for the fertilization procedure, the incubation and the aquaria water was directly supplied by a stationary lake water pipe out of 60 m depth in the lake and was used in an unmodified way for the entire study (no additional temperature adjustment or filtration was needed in the lab). 2.2. Experimental design From fertilization until hatch, brown trout embryos were exposed once or several times for 2 h to a solvent control (Ctrl), 3.8 ng/L 17-estradiol (E2, 98% purity, E8875, Sigma–Aldrich, Buchs, Switzerland) or 38.0 ng/L E2 in aerated 10-L-aquaria filled with cold lake water (6.1 ◦ C). Stock solution of 1 mg/mL E2 in ethanol (99.8% purity, Sigma–Aldrich, Buchs, Switzerland) was diluted in lake water (final concentration of ethanol in the aquaria water
