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Zebrafish Danio rerio and roach Rutilus rutilus: Two species suitable for evaluating effects of endocrine disrupting chemicals?
Abstract
Morphological studies of gonads from roach (Rutilus rutilus) in a small Swedish lake were performed. The lake is a potential recipient for drainage water from a refuse dumping area. All fishes were macroscopically identified as males, but histological examination of the gonads showed a high incidence of intersex. This high incidence might be caused by endocrine disrupting chemicals. The relationships between exposure to endocrine disrupters and intersex observations in wild fish is unknown and the effect of endocrine disrupters on zebrafish (Danio rerio) were evaluated in order to determine whether they might serve as a model laboratory species for effects of endocrine disrupting chemicals in wild fish. Zebrafish were exposed from pre-blastula stage until sexual maturation to either standardised water (controls), 17a-methyltestosterone (1, 10 g l-1) or 17b-estradiol (1, 10 g l-1). Exposure to both concentrations of 17bestradiol caused a significant feminisation, while a significant increase in the proportions of males was found after exposure to 1 mg 17a-methyltestosterone l-1. Furthermore, exposure to 1 and 10 mg 17a-methyltestosterone l-1 caused development of intersex individuals. Additionally, sexually mature male zebrafish were exposed to 1 g 17b-estradiol l-1. After 10 weeks of exposure the fish were transferred to non-contaminated water for two weeks. The control group was kept in standardised water for 12 weeks. At termination of the experiment, measurement of whole-body vitellogenin concentrations were performed. Exposure to 17b-estradiol caused a significant increase in whole body vitellogenin concentrations. After 2 weeks in water without addition of 17b-estradiol, the vitellogenin concentration was reduced by approximately 50%. Roach and zebrafish may share properties which makes these species suitable for further comparisons to evaluate effects of endocrine disrupting chemicals as well as domestic and industrial effluents containing hormone-like substances.
... Domestic sewage effluents have been reported to contain estrogenic compounds, both the endogenous hormone estradiol (E2) and the metabolites, estrone (E1) and estriol (E3) as well as the active compound in contraceptives, the synthetic hormone ethinylestradiol (EE2) (Desbrow et al., 1998;Belfroid et al., 1999;Larsson et al., 1999;Ternes et al., 1999;Körner et al., 2000; Kuch and Ballschmitter, 2000;Spengler et al., 2001). Apart from reports from the UK, intersexuality in roach populations has also been reported from other European countries, including Finland, Sweden and Denmark (Wiklund et al., 1996;Andersen et al., 2001;Christiansen et al., 2000). Intersex in wild fish has also been described in a number of other species, such as barbel (Barbus plebejus), bream (Abramis brama), gudgeon (Gobio gobio), flounder (Platichthys flesus), and carp (Cyprinus carpio) ( Vigano et al., 2001;Vethaak et al., 2002; Van Aerle et al., 2001; Hashimoto et al., 2000;Matthiessen et al., 1998;Allen et al., 1999;Sole et al., 2002). ...
... Intersex has also been observed in other fish species after exposure to MT Rinchard et al., 1999;Al-ablani and Phelps, 1997). Previous studies on zebrafish have shown that exposure to 1000 and 10,000 ng MT/l from fertilization to sexual maturation resulted in intersex fish ( Andersen et al., 2001). Furthermore, a shift in gonadal sex ratios, with masculinization taking place in fish exposed to 1000 ng/l and feminization of fish at 10,000 ng/l was reported ( Andersen et al., 2001). ...
... Previous studies on zebrafish have shown that exposure to 1000 and 10,000 ng MT/l from fertilization to sexual maturation resulted in intersex fish ( Andersen et al., 2001). Furthermore, a shift in gonadal sex ratios, with masculinization taking place in fish exposed to 1000 ng/l and feminization of fish at 10,000 ng/l was reported ( Andersen et al., 2001). Female-biased sex ratios in fish after exposure to MT, being known as a paradoxical feminization, have been reported from other studies ( Rinchard et al., 1999;). ...
In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.
... Fish reared at high population density tend to become males (Liew et al., 2012;Sfakianakis 2012;Ribas et al., 2017). Treatment with 17β-estradiol causes fish feminization (Andersen et al., 2001). Higher food intake also promotes female development (Lawrence et al., 2008). ...
Zebrafish is a popular research model; but its mechanism of sex determination is unclear and the sex of juvenile fish cannot be distinguished. To obtain fish with defined sex, we crossed domesticated zebrafish with the Nadia strain that has a female-dominant W segment. These fish were placed on a ziwi:GFP background to facilitate sorting of fluorescent germ cells for transcriptomic analysis. We analyzed the transcriptomes of germ cells at 10–14 days postfertilization (dpf), when sex dimorphic changes started to appear. Gene ontology showed that genes upregulated in the 10-dpf presumptive females are involved in cell cycles. This correlates with our detection of increased germ cell numbers and proliferation. We also detected upregulation of meiotic genes in the presumptive females at 14 dpf. Disruption of a meiotic gene, sycp3, resulted in sex reversal to infertile males. The germ cells of sycp3 mutants could not reach diplotene and underwent apoptosis. Preventing apoptosis by disrupting tp53 restored female characteristics in sycp3 mutants, demonstrating that adequate germ cells are required for female development. Thus, our transcriptome and gene mutation demonstrate that initial germ cell proliferation followed by meiosis is the hallmark of female differentiation in zebrafish.
... The sex ratio expected for zebrafish reared under normal laboratory conditions is theoretically 1 female: 1 male (Takahashi, 1977), but it has been reported that it is generally around 40: 60 (female to male), and therefore with a small predominance of males (Ankley and Johnson, 2004). However, different studies show how sex ratio can be extremely variable in this species, such as: 31: 69 , 32: 68 (Andersen et al., 2001), 40: 60 (Mikula et al., 2009), 72: 38 (Andersen et al., 2005 and 67: 33 (Örn et al., 2003, 2006b) female to male. The use of different laboratory strains, which differ in their genetic background (Brown et al., 2012a;Guryev et al., 2006), have been take into account to explain the natural variation of zebrafish. ...
Zebrafish (Danio rerio) is extensively used in research; however the mechanisms that control this species sex determination are still poorly understood. In the latest decades, it has been established that zebrafish sex is determined by genetic factors on a polygenic basis, as various candidate genes with sex dimorphic expression, as well sex-linked loci have been identified in different zebrafish strains. However, it has been evidenced that sex determination in this species is also influenced by environmental factors. For instance, temperature can have a crucial role in zebrafish sex determination. Likewise, the exposure to endocrine disrupting chemicals (EDCs), the most studied zebrafish sex changing factor, can strongly influence the course of sex differentiation and unbalance the sex ratio of zebrafish populations. Despite this, so far the influence of environmental factors is still less understood and only few studies have addressed this topic. Therefore, this review intends to gather current knowledge on the environmental factors involved in sex determination of zebrafish and identify important gaps in this research area. Briefly, the current understanding on zebrafish sex related genetics is also addressed.
... Considering the threat to natural populations, there is a need to develop standard testing methods to ascertain impacts of EDSs in fish [1,15]. While some whole animal lifecycle bioassays have been developed for freshwater fish such as fathead minnows (Pimephales promelas) [16][17][18], Japanese medaka (Oryzias latipes) [19,20], and zebrafish (Danio rerio) [21], few, with the exception of the sheepshead minnow (Cyprinodon variegatus) [22] and recently with the mummichog (Fundulus heteroclitus) [14,23], have been developed for marine and estuarine fish. ...
To evaluate the use of morphological abnormalities for standard testing of endocrine-disrupting substances (EDS), we tested the hypothesis that developmental abnormalities are a sensitive indicator of exposure to waterborne estrogenic and antiestrogenic EDS during embryonic, larval, and juvenile stages in the common estuarine killifish, the mummichog (Pisces: Cyprinodontidae). Static exposures with daily renewal were carried out with 10 to 10,000 ng/L of the estrogen agonist 17alpha-ethynylestradiol (EE2) or antagonist ZM189,154 (ZM) for the first 25 or 60 d of life. Incidence of skeletal abnormalities (scoliosis, lordosis, head, craniofacial, jaw, fin) and soft tissue abnormality (anal swelling) were significantly increased by EE2 but only at high concentrations (1,000 or 10,000 ng/L). Sixty-day exposure produced more severe abnormalities than 25-d exposure and in a higher proportion of fish. Within the longer exposure, 10,000 ng/L EE2 produced more abnormal fish than 1,000 ng/L (100% vs 51.6%) and more abnormalities per abnormal fish (5.73 vs 1.47). Fish reared to 12 months in clean water after exposure for 60 d to 10,000 ng/L EE2 survived at a lower rate than controls, retained abnormalities with the exception of anal swelling and, like fish exposed to other concentrations of EE2 and ZM, showed increased weight at length at 6 and 12 months. Sixty-day exposure to ZM increased the incidence of scoliosis (1,000 ng/L) but decreased the overall incidences of abnormal fish and lordosis (10 and 10,000 ng/L). No impacts of EE2 or ZM were observed before hatch, and clearing and staining of larvae demonstrated that expression of vertebral abnormalities coincided temporally with ossification. We conclude that morphological abnormalities in mummichogs are not a sensitive indicator of exposure to estrogenic or antiestrogenic waterborne EDSs at environmentally. relevant concentrations.
... The incidence of intersex has been attributed to the presence of xenoestrogens in the sewage effluent. Intersex in wild roach has also been found in Scandinavian countries (Wiklund et al., 1996;Andersen et al., 2001;Bjerregaard et al., 2005). ...
... Roach are a species that has been widely studied for intersex in Europe, i.e. England (Jobling et al., 1998(Jobling et al., , 2006Minier et al., 2000;Nolan et al., 2001), France (Maltret-Geraudie et al., 2008;Minier et al., 2000), Sweden (Andersen et al., 2001), Denmark (Bjerregaarda et al., 2006) and Ireland (McGee et al., 2012). This species was specifically studied at a reference site in Normandy (France) that was thought to be free of anthropogenic influences (industrial, agricultural or municipal effluents). ...
... While certain authors observed a dominance of male zebrafish in control groups of their experiments (e.g. 69% and 58%, Brion et al. 2004, or 68%, Andersen et al. 2001), other authors reported a balanced (i.e. approximately 1 : 1) sex ratio (Maack and Segner 2004; Drastichová et al. 2005). ...
The aim of the study was to evaluate the xeno-oestrogenic potential of propylparaben in vivo using zebrafish (Danio rerio). Experimental juvenile zebrafish (20 days post hatching) were fed a feed containing 500, 1000, or 2000 mg·kg -1 of propylparaben, fish in a positive control group were given a feed treated with 20 mg·kg -1 of 17β-oestradiol, and the control fish were given the feed free of either tested substance. The exposure of fish to propylparaben did not affect vitellogenesis after 20 days exposure but seemed to influence the sex differentiation processes, as evidenced by a sex ratio significantly skewed towards females in the group fed 500 mg·kg -1 of propylparaben following 45 days of exposure. The potential of the fish to respond to oestrogenic stimulation was confirmed, since significantly higher vitellogenin concentrations were detected in the fish from the positive control group.
... Most previous studies have suggested that endosulfan could seriously impact fish population health by influencing characteristics such as the growth and development, mobility, foraging success, and reproductive performance of exposed individuals [17,18]. The serum triiodothyronine, thyroxine, prolactin, and insulin levels were found to be increased, and the cortisol level in serum was reduced in Sarotherodon mossambicus [19] exposed to a sublethal (0.001 mg/L) concentration of endosulfan [20]. The embryos and larvae of zebrafish exposed to endosulfan exhibited an abnormal response to touching, suggesting that endosulfan is developmentally toxic to zebrafish [21,22]. ...
Because of persistent organic pollution in aquatic environments, the widely used organochlorine pesticide endosulfan, which is a potential endocrine disruptor, is expected to pose a significant risk to aquatic organisms. In the present study, we explored the potential endocrine-disrupting risk of β-endosulfan by investigating its effect on the growth, reproduction, plasma vitellogenin, and organ histology of adult zebrafish. We found that, although β-endosulfan did not significantly affect the growth of zebrafish, it greatly decreased the hatching rate, even at a concentration as low as 10 ng/L. Interestingly, the decrease of the hatching rate was highly correlated with pathological alterations of the testes. Additionally, the values of the gonadosomatic index were significantly reduced in female zebrafish treated with 200 ng/L β-endosulfan, which was also closely associated with ovarian histological changes. More importantly, a significant increase in the level of vitellogenin was observed in all male fish treated with β-endosulfan. Based on these findings, we conclude that β-endosulfan severely affects the reproductive function of zebrafish and the synthesis of vitellogenin in the liver, and thus, β-endosulfan has a serious endocrine disruption function in zebrafish.
... Over the last decade, concern has been raised about the potential effects of endocrine-disrupting chemicals (EDCs) on the development and reproduction of humans and wildlife (Colborn et al. 1996). Effects of EDCs in fish include reduced fertility (decreased sperm number and quality, or egg number), induction of vitellogenin (VTG) in males and juveniles, and effects on the development of the gonads (Billard et al. 1981; Andersen et al. 2001; Aguayo et al. 2004). The Office of Research and Development of the United States Environmental Protection Agency (US EPA) has identified EDCs issues as one of six high-priority research areas (US EPA 1996; Kavlock et al. 1996). ...
Present in the excrement of humans and animals, 17beta-estradiol (E(2)) has been detected in the aquatic environment in a range from several nanograms to several hundred nanograms per liter. In this study, the sensitivities of rare minnows during different life stages to E(2) at environmentally relevant (5, 25, and 100 ng l(-1)) and high (1000 ng l(-1)) concentrations were compared using vitellogenin (VTG) and gonad development as biomarkers under semistatic conditions. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed; the results indicated that the lowest observed effective concentration for VTG induction was 25 ng l(-1) E(2) in the adult stage, but 100 ng l(-1) E(2) in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile fish were transferred to clean water until gonad maturation. No significant difference in VTG induction was found between the exposure and control groups in the adults. However, a markedly increased proportion of females and appearance of hermaphrodism were observed in the juvenile-stage group exposed to 25 ng l(-1) E(2). These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E(2). The juvenile stage may be the critical period of gonad development. Sex ratio could be a sensitive biomarker indicating exposure to xenoestrogens in early-life-stage subchronic exposure tests. The results of this study provide useful information for selecting sensitive biomarkers properly in aquatic toxicology testing.
... genic and anti-androgenic effects are known as well. Effects of endocrine disrupters in fish include reduced fertility (decreased sperm number and quality, or egg number), induction of the synthesis of the yolk precursor protein vitellogenin (VTG) in males and juveniles and effects on the development of the gonads (Billard et al., 1981; Andersen et al., 2001; Orn et al., 2000). ¨ Endocrine disrupters are effective at sub-lethal concentrations (Stahlschmidt-Allner et al., 1997) and it is, therefore, not possible to detect effects of such compounds using existing standard tests with endpoints such as mortality. ...
To determine the critical stage of zebrafish development where exposure to xenoestrogens can affect sex ratio and vitellogenin induction, zebrafish (Danio rerio) were exposed to 17alpha-ethinylestradiol (actual concentration 15.4+/-1.4 ng EE2/l) during early development: from fertilisation to hatch; hatch to 10 days post hatch (dph); 10-20 dph; 20-30 dph; 20-40 dph; 20-60 dph; fertilisation to 25 dph; or hatch to 60 dph. Vitellogenin was measured in whole body homogenate 30 dph by ELISA and sex ratio was determined 60 dph by histological examination of the gonads. All exposure periods significantly induced vitellogenin synthesis and affected the sex differentiation leading to development of ovo-testis or complete feminisation of the exposed fish depending on exposure period. Complete sex reversal was obtained in groups exposed from 20 to 60 dph and hatch to 60 dph. The half-life for degradation of vitellogenin was calculated. Juvenile zebrafish were exposed to 15.4+/-1.4 ng EE2/l (actual concentration) from fertilisation to 25 dph and transferred to clean water, after which weekly measurements of vitellogenin concentration in whole body homogenate were performed until day 46 post hatch. The half-life of vitellogenin was 2.4 days.
... Laboratory-held species also vary in their responses; sunshine bass (Morone sp.) respond to sewage exposure by increasing liver vitellogenin (VTG) production while mummichog (Fundulus heteroclitus) do not [5]. Development of partial and full life-cycle fish tests for EDS testing has primarily focused on freshwater species such as fathead minnow, Pimephales pro- melas [1,15,17], Japanese medaka, Oryzias latipes [13,18], and zebrafish, Danio rerio [19,20]. With the exception of tests with the sheepshead minnow [21,22], there is a paucity of methods suitable for estuarine and saltwater species. ...
We have developed a short-term gonadal recrudescence test with the estuarine mummichog (Fundulus heteroclitus) and determined endocrine end points sensitive to a strong estrogen agonist (ethynylestradiol; EE2) and an antiestrogen (ZM 189,154; ZM) at concentrations of 0 to 1,000 ng/L in three separate experiments. A protocol was developed to ensure a year-round supply of recrudescing fish. A protocol for determining steroid production (testosterone and 11-ketotestosterone [11-KT] in incubated testes tissue and testosterone and 17-estradiol [E2] in incubated prematurational follicles) was optimized. Recrudescing fish (males, gonadosomatic index = 2%; females = 10%) were exposed to graded doses of EE2 or ZM for 7 to 15 d using a static daily-renewal protocol. At high EE2 (>250 ng/L), the effect on males was depression of androgen steroidogenesis and plasma steroid levels. In females, high EE2 depressed gonadal production and circulating E2 levels; however, EE2 concentrations <100 ng/L caused increased gonadal production and plasma E2. Low ZM (<100 ng/L) had little effect on male and female fish, while higher concentrations (>250 ng/L) increased E2 and 11-KT production while decreasing plasma 11-KT and E2 (1,000 ng/L only). Male and female plasma vitellogenin responded in a concentration-dependent fashion to EE2 with no effect by ZM. The low observable effect concentrations for the endocrine parameters were 1 ng/L for EE2 and 250 ng/L for ZM. The bioassay and results encompassing the environmentally relevant exposure range (1-100 ng/L) will be useful for assessing effects of endocrine-active contaminants in estuarine environments.
... It is well known that chemicals with different mode of action can affect fish. Examples are: androgenic (Andersen et al., 2001Andersen et al., , 2006 Ankley et al., 2001; Zerulla et al., 2002; Örn et al., 2003; Pawlowski et al., 2004; Sone et al., 2005) and estrogenic (Sumpter and Jobling, 1995; Routledge et al., 1998; Akerblom et al., 2000; Van der Ven et al., 2003; Brion et al., 2004) as well as anti-androgenic (Bayley et al., 2003; Kinnberg and Toft, 2003) and anti-estrogenic (Smeets et al., 1999; Panter et al., 2002; Andersen et al., 2005) effects. In the present study, three estrogens and one androgen were chosen as test compounds; the natural estrogens, estrone (E1), 17β-estradiol (E2) and estriol (E3) are mainly produced and excreted by female mammalians and they are suspected to play a key role in the field observations of elevated vitellogenin concentrations and intersex in fish, because they can be found in concentrations which have caused similar effects in laboratory studies (Routledge et al., 1998; Metcalfe et al., 2001; Bjerregaard et al., 2006; Van den Belt et al., 2004). ...
Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.
Severely skewed sex ratios in zebrafish stocks can pose significant hurdles for line propagation and sperm cryopreservation. To overcome female-biased sex ratios in stocks derived from imported sperm samples, the Zebrafish International Resource Center has implemented routine supplementation of larval food with 17α-methyltestosterone to skew gonadal sex differentiation toward masculinization. Resulting stocks averaged 80% males.
This work aimed to investigate the effects of the endocrine disruptors 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) on Nile tilapia (Oreochromis niloticus) development, emphasizing the zootechnical and morphological aspects. The concentrations of E2 and EE2 tested were 250, 500, and 1,000 μg·L⁻¹. The evaluated compounds were capable of producing intersex individuals and causing zootechnical damage in Nile tilapia, with a significant decrease in the condition factor as the concentrations increased. Besides, these concentrations were also able to induce the development of morphological anomalies without any significant difference between them. E2 and EE2 exposure were shown to be lethal for Nile tilapia larvae, having no effect on the incubation time and the percentage of larvae hatching. Morphological anomalies such as head shape malformation, oral malformation, operculum malformation, belly retraction, distended abdomen with fluid accumulation (ascites), exophthalmos, signs of bleeding in the belly, and curved pectoral fin radii, were also observed, which impaired the fish development.
Keywords:
endocrine disruptors; Nile tilapia (Oreochromis niloticus); zootechnical aspects; morphological aspects
We have developed a short-term gonadal recrudescence test with the estuarine mummichog (Fundulus heteroclitus) and determined endocrine end points sensitive to a strong estrogen agonist (ethynylestradiol; EE(2)) and an antiestrogen (ZM 189,154; ZM) at concentrations of 0 to 1,000 ng/L in three separate experiments. A protocol was developed to ensure a year-round supply of recrudescing fish. A protocol for determining steroid production (testosterone and 11-ketotestosterone [11-KT] in incubated testes tissue and testosterone and 17-estradiol [E(2)] in incubated prematurational follicles) was optimized. Recrudescing fish (mates, gonadosomatic index = 2%; females = 10%) were exposed to graded doses of EE(2) or ZM for 7 to 15 d using a static daily-renewal protocol. At high EE(2) (>250 ng/L), the effect on males was depression of androgen steroidogenesis and plasma steroid levels. In females, high EE(2) depressed gonadal production and circulating E(2) levels; however, EE(2) concentrations <100 ng/L caused increased gonadal production and plasma E(2). Low ZM (<100 ng/L) had little effect on male and female fish, while higher concentrations (>250 ng/L) increased E(2) and 11-KT production while decreasing plasma 11-KT and E2 (1,000 ng/L only). Male and female plasma vitellogenin responded in a concentration-dependent fashion to EE(2) with no effect by ZM. The low observable effect concentrations for the endocrine parameters were 1 ng/L for EE(2) and 250 ng/L for ZM. The bioassay and results encompassing the environmentally relevant exposure range (1-100 ng/L) will be useful for assessing effects of endocrine-active contaminants in estuarine environments.
In the present study, the effects of the anti-estrogen ZM 189,156 and the aromatase inhibitor fadrozole were evaluated in
a 40-day juvenile assay developed for screening of endocrine disrupting chemicals. Juvenile zebrafish were exposed from 20
to 60days post hatch (dph) to ZM 189, 154 (100μgl−1 and 200μgl−1) and fadrozole (10, 32, and 100μgl−1). VTG concentrations were measured at 38dph by the use of a direct non-competitive sandwich ELISA and sex ratios were determined
at 60dph by histological examination of the gonads. A small but significant increase in VTG concentrations was observed in
fish exposed to ZM 189, 156 (100 and 200μgl−1) and fadrozole (10 and 100μgl−1) compared to control groups. In fish exposed to ZM 189, 156 and fadrozole, the percentage of females declined and the number
of undifferentiated fish increased. These findings show that exposure of juvenile zebrafish to an aromatase inhibitor or an
anti-estrogen during early development inhibits differentiation and development of female gonads. The data presented, furthermore,
show that the 40-day juvenile assay may be suitable for screening endocrine disrupting chemicals acting as anti-estrogens
and aromatase inhibitors.
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The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
This study investigated the suitability of juvenile three-spined sticklebacks, Gasterosteus aculeatus L., for detecting both androgen- and oestrogen-induced endocrine disruption. The investigated endpoints were kidney hypertrophy and the induction of the protein markers spiggin and vitellogenin. Juveniles were exposed to steroid hormones 17 beta-oestradiol (E2: nominal 0.01, 1.0 and 10 microg/L), 17 alpha-ethinylestradiol (EE2: nominal 0.05 microg/L) and 17 alpha-methyltestosterone (MT: nominal 1.0 microg/L) from the day of hatching until the termination of the experiments between 39 and 58 days after hatching. E2 (10 microg/L) and MT were applied during different time windows: (a) 14 days after hatching only and (b) continuously with start 14 days after hatching. Kidney hypertrophy is an androgen-dependent secondary sexual character in adult male sticklebacks and corresponds to the production of the glue protein spiggin during the breeding season. The kidneys were hypertrophied and spiggin levels were elevated in juvenile sticklebacks after treatment with MT. Paradoxically, slightly elevated spiggin levels and kidney hypertrophy were observed also in fish treated with high dose E2. Levels of vitellogenin, the oestrogen-inducible yolk precursor protein, were elevated in juvenile sticklebacks after E2 medium and high dose and EE2 treatment. The tested endpoints are suitable for the study of endocrine disruption in juvenile sticklebacks, a fish species that is easy to handle in laboratory and relevant for temperate geographical regions.
Juvenile three-spined stickleback (Gasterosteus aculeatus L.) is introduced as a unique model organism for both androgenic and oestrogenic endocrine action. Intersex is often used as an indicator for disruption of sexual differentiation in fish exposed to different kinds of effluents from human activities. In wild fish it has exclusively been reported in terms of feminisation due to xenoestrogens in the environment. The assumption that the intersex individuals are feminised genetic males can only be proven by genetic sex identification of the intersexual individuals. Intersex and gonadal sex reversal were induced in three-spined sticklebacks by treatment with natural and synthetic steroid hormones. Juvenile sticklebacks were exposed to three nominal concentrations of 17 beta-oestradiol (E2); i.e. 0.01, 1.0 and 10.0 microg/L; which were administered to the water either continuously from hatching to the end of the experiment (39-58 days post hatch), during the first 2 weeks after hatching only, from 14 days after hatching onwards, or during the chorionated embryo stage until hatching. Other groups were exposed to 17 alpha-ethinylestradiol (EE2) at 0.05 microg/L and 17 alpha-methyltestosterone (MT) at 1.0 microg/L (nominal concentrations). MT was applied continuously, during the first 2 weeks post hatch only, or from 14 days after hatching onwards. Gonad histology was examined and the genetic sex was identified with male sex-linked PCR markers. Treatment with oestrogens caused feminisation at the two highest E2 concentrations and with EE2. Exposure to E2 before hatching had no effect. Intersexual individuals from oestrogen treatments were genetic males. The genetic sex marker identified apparent total reversal of the gonad type of genetic males. Treatment with MT did not reveal a clear picture, since intersex was observed in both genetic females and males. MT also caused severe testis abnormalities, mainly the development of large branched cavities with unidentified origin. The process of sex differentiation is most sensitive to the influence of external steroids during the first 2 weeks after hatching. A lower incidence of intersex could also be induced in sticklebacks exposed from 14 days after hatching by E2 treatment, but not with MT. The combination of gonad histopathology with genetic sex identification in juvenile sticklebacks is suggested as a tool for detecting endocrine disruption in laboratory studies, and might become very useful in field surveys.
We tested the hypothesis that gross morphological abnormalities are a sensitive indicator of exposure to waterborne androgenic and anti-androgenic compounds during embryonic, larval and juvenile stages of development in the common estuarine killifish, the mummichog (Fundulus heteroclitus; Pisces: Cyprinodontidae). Static exposures with daily renewal were carried out with 10-100,000 ng/L of the androgen agonist, 17alpha-methyltestosterone (MT), or the androgen antagonist, cyproterone acetate (CA), for 60 days post-fertilization (PF) in duplicate exposures. Measured concentrations were 78.4-155.8% of nominal concentrations for MT and 13.5-168.1% for CA. No dose-related or consistent effects of MT or CA were observed before hatch. In 60 days PF juveniles, incidence of skeletal abnormalities (scoliosis, lordosis, head, facial and fin), soft tissue abnormality (anal swelling) and hemorrhaging were significantly increased by MT but only at high concentrations (> or =1000 ng/L). The 10,000 and 100,000 ng/L concentrations of MT produced a wider range of abnormalities than 1000ng/L. Over 90% of fish exposed to 10,000 or 100,000 ng/L were abnormal with an average of over 3.5 abnormalities per fish. CA did not increase the incidence of any type of abnormality. Survival of juveniles to the end of the exposure was reduced by MT at concentrations of 1000 ng/L and greater in the first experiment and at concentrations of 10,000 ng/L and greater in the second experiment. Juvenile length was reduced by high concentrations of MT (> or =10,000 ng/L) in the first experiment and by most concentrations in the second experiment. We conclude that morphological abnormalities in early-life stages of mummichogs are not a sensitive indicator of exposure to androgenic or anti-androgenic waterborne EDSs at environmentally relevant concentrations.
To evaluate the use of morphological abnormalities for standard testing of endocrine-disrupting substances (EDS), we tested the hypothesis that developmental abnormalities are a sensitive indicator of exposure to waterborne estrogenic and antiestrogenic EDS during embryonic, larval, and juvenile stages in the common estuarine killifish, the mummichog (Pisces: Cyprinodontidae). Static exposures with daily renewal were carried out with 10 to 10,000 ng/L of the estrogen agonist 17alpha-ethynylestradiol (EE2) or antagonist ZM189,154 (ZM) for the first 25 or 60 d of life. Incidence of skeletal abnormalities (scoliosis, lordosis, head, craniofacial, jaw, fin) and soft tissue abnormality (anal swelling) were significantly increased by EE2 but only at high concentrations (1,000 or 10,000 ng/L). Sixty-day exposure produced more severe abnormalities than 25-d exposure and in a higher proportion of fish. Within the longer exposure, 10,000 ng/L EE2 produced more abnormal fish than 1,000 ng/L (100% vs 51.6%) and more abnormalities per abnormal fish (5.73 vs 1.47). Fish reared to 12 months in clean water after exposure for 60 d to 10,000 ng/L EE2 survived at a lower rate than controls, retained abnormalities with the exception of anal swelling and, like fish exposed to other concentrations of EE2 and ZM, showed increased weight at length at 6 and 12 months. Sixty-day exposure to ZM increased the incidence of scoliosis (1,000 ng/L) but decreased the overall incidences of abnormal fish and lordosis (10 and 10,000 ng/L). No impacts of EE2 or ZM were observed before hatch, and clearing and staining of larvae demonstrated that expression of vertebral abnormalities coincided temporally with ossification. We conclude that morphological abnormalities in mummichogs are not a sensitive indicator of exposure to estrogenic or antiestrogenic waterborne EDSs at environmentally relevant concentrations.
Single pairs of zebrafish, Brachydanio rerio, 12 months old, spawned at an interval of 1.9 days when the male and female were left continuously together. Three months later the spawning interval had increased to 2.7 days, suggesting that age is a factor in determining the spawning cycle. Hard water had no effect on spawning cycle in these experiments, nor did grouping the fish with three males for each female. An experiment was done in which prolongation of the spawning cycle was enforced by separating the male from the female for random intervals of 2-9 days. In 93% of the trials spawning occurred on the morning following reintroduction of the male after a 2-day separation. Similar results were obtained upon reintroduction of the male after 3-9 days of separation. The greatest number of eggs per day can be obtained by leaving the pair continuously together. In two experiments in which this was done the mean number of eggs produced per day per pair was 23.1 and 60.4. In the experiment where male and female were separated for 2-9 days, and then spawning was allowed to occur, egg production varied from 45 eggs per day per pair with 2 days of separation to 10 eggs per day per pair with 9 days separation. Evidence was obtained that egg development is triggered by interaction with the male. Eggs usually could not be stripped from the female without the male having been present. Introduction of the male for 7 hours at the end of one day enabled eggs to be stripped from the female the next morning. The precise stimulus for egg development was not determined, but it is suggested that it may be the chasing of the female by the male, behavior which goes on almost continuously when the two are together.
Gonadal impairment in roach Rutilus rutilus from Finnish coastal waters was studied during 1987-1995. The disease was observed in high prevalence (>10%, for roach in length class 25-30 cm) in roach from 4 sites in the Archipelago Sea, from one site influenced by cooling water discharge from a nuclear power plant in Gulf of Bothnia, and in roach from one site close to a metal processing plant in the northern Gulf of Bothnia. The sites in the Archipelago Sea were not directly affected by industrial pollution. The disease was most prevalent in large (old) female roach, and it was characterized by degeneration of the ovaries ranging from weak tissue changes in one ovary to complete absence or destruction of both ovaries. Oocytes, in affected gonads, were frequently infected by a microsporidian parasite Pleistophora mirandellae. In males atretic testes were never observed and only small cysts, containing Fl mirandellae, were detected. In several affected roach, both male and female reproductive tissues were observed. The observed pathological changes in the ovaries of roach are suggested to be caused by P. mirandellae.
To analyse a recruitment failure in a roach (Rutilus rutilus) population exposed to cooling water from a Swedish nuclear power plant, histopathological examinations were made on female gonads. Several disorders were documented, corresponding to effects previously seen in Lithuanian, Moldavian and Russian heated waters, The relative gonad size (GSI) was generally lower in the exposed population. Oocyte degeneration was observed in about 50% of the females, potentially seriously reducing their reproductive capacity. The gameto- and gonadogeneses also were clearly affected and generally occurred at a faster rate. Sexual maturity was earlier and was accompanied by an increased occurrence of asynchionic gonad and oocyte growth. A normal gonad development should pass through different stages, coupled to the seasons, and the difference between individuals in a population should be small. However, in some exposed fish, the sequence of seasonal development was disturbed and a simultaneous, intensive, growth of sex cells in different stages of development could be seen. An increase of variation in gonad development both within, as well as between, fish thus was documented. Conclusively, the histopathological analysis provided evidence that the observed recruitment deficit could be related to gonad failure.
High temperature in Swedish and Lithuanian thermal effluent areas influenced gametogenesis of female perch Perca fluviatilis, roach Rutilus rutilus and pike Esox lucius negatively, indicating reduced reproductive capacity. Oocyte atresia started during vitellogenesis in autumn, and was often followed by asynchronous egg cell development. Among other anomalies, multi-nucleus oocytes and hermaphroditism were observed. No significant impact was seen in silver bream Blicca bjoerkna. Ruffe Gymnocephalus cernuus reacted by a tendency to produce an additional mature oocyte generation during the spawning period. Ovaries in roach from coastal areas were often infected by a microsporidian parasite Pleistophora mirandellae, causing severe damage to the gonad. Parasites were also detected in pike, but neither in perch nor in lake populations of roach. Fish living in open coastal environments did not avoid impact by moving out of the heated areas. There seems to be a conflict in some temperate fish between temperature preference behaviour and safeguarding normal reproduction.
The liver is the major organ of detoxification in all vertebrates and would therefore be expected to play a major role in determining the sensitivity of an organism to a particular pollutant. In fish it is particularly rich in lipids and it is therefore not surprising to find that it is also a major site of bioaccumulation for many organic pollutants (see Chapter 3). Such bioaccumulation increases the risk both of morphological damage to the liver, and of disruption to the activities of detoxifying enzymes. These changes may in turn alter the rate at which further accumulations of the toxin can be deactivated and removed from the organism. This could result in synergisms between different pollutants if the disruption of the deactivating systems caused by one pollutant results in failure to detoxify the second. The hepatic enzymes used for detoxifying xenobiotics are in fact closely related to the enzymes that are used for both synthesis and deactivation of other chemicals naturally present in the body. P450 and conjugating enzymes, for example, play a major role in the deactivation of steroid hormones and if these enzymes are preferentially used to metabolise a pollutant, the hormones may be less rapidly deactivated resulting in a higher plasma concentration. They are also essential for the synthesis of steroid hormones by the interrenal and gonads. The situation is further complicated since the liver is both the target tissue and site of deactivation of the ovarian steroid estradiol. As a target tissue it has estrogen receptors which specifically induce synthesis of the yolk protein vitellogenin. The balance between binding to such receptors and deactivation could be affected by changes both in the deactivating enzymes and in the structural integrity of the liver and its constituent cells.
The estrogenicity of several xenobiotics was evaluated using in vivo vitellogenin (Vtg) synthesis in immature rainbow trout as a biomarker. 17β-estradiol, DES and ethinyl estradiol were tested as positive controls. The xenobiotic compounds tested were technical nonylphenol, bisphenol A, butylbenzylphthalate (BBP) and dibutylphthalate (DBF). Measurements of the Vtg concentration was performed with a direct sandwich ELISA. 17β-estradiol, DES and ethinyl estradiol caused up to 100 000-fold increases in the Vtg-levels. Nonylphenol and bisphenol A had the highest estrogenic potency of the xenobiotics increasing the vitellogenin level approximately 300-fold while BBP was only weakly estrogenic, increasing the concentration about 3 times. DPB did not raise the vitellogenin contration above the detection limit.
The use of all-male populations increases the efficiency and feasibility of tilapia aquaculture. The objective of this study was to determine the efficacy of a short-term immersion procedure for masculinizing Nile tilapia (Oreochromis niloticus). Two synthetic androgens were evaluated: 17α-methyldihydrotestosterone (MDHT) and 17α-methyltestosterone (MT). Exposure (3h) on 10 and again on 13 days post-fertilization to MDHT at 500μg/1 successfully masculinized fry in all experiments, resulting in 100, 94 and 83±2% males in Experiments 1, 2 and 3, respectively. Immersions in MDHT or MT at 100μg/1 resulted in significantly skewed sex ratios in Experiments 1 and 3 (MT resulted in 73 and 83±3% males; and MDHT resulted in 72 and 91±1% males) but not in Experiment 2. Immersion in MT at 500μg/1 only caused masculinization in Experiment 3. Although further research and refinement is needed, immersion of Nile tilapia in MDHT may provide a practical alternative to the use of steroid-treated feed. Furthermore, when compared with current techniques for steroid-induced sex inversion of tilapia, short-term immersion reduces the period of time that workers are exposed to anabolic steroids.
The sex of gynogenetic rainbow trout was reversed to produce XX males by using two steroids, 17α-methyltestosterone (MT) and 11 β-hydroxyandrostenedione (OHA). Fish were exposed to either single or multiple doses of steroids during various times around the period of hatching to determine the labile period for effective sex reversal. Steroids were administered either by immersion (400 μg/ 1 for 2 h) or a combination of immersion plus feeding (3 mg/kg diet for 60 days) to determine if males with intact sperm ducts could be produced. Immersion in MT resulted in varying degrees of masculinization while immersion plus feeding produced nearly 100% male populations. The most effective period for steroid immersion was 1 week after the time when one-half of the fish had hatched. Multiple immersions in MT failed to increase masculinizing effects. Immersion in OHA caused only low rates of masculinization, while immersion plus feeding resulted in 70% male populations. Males produced through both immersion and feeding of MT generally did not develop sperm ducts; whereas animals treated by immersion alone in MT, or those produced with OHA, tended to be functional. Although final yields for sex-reversed males following gynogenesis were low, 1.5 and 1.6% for the two groups respectively, cryopreserved semen from these males can be used to produce all-female offspring for years. Cryopreserved semen from functional males in this study produced 100% female populations.
Methods are described for controlling sex differentiation in the coho salmon, Oncorhynchus kisutch. Eyed eggs and alevins were immersed in solutions of estradiol-17β and 17α-methyltestosterone and fed these steroids in the diet for a period of 10 weeks postswim-up. In seven out of 10 groups that received estradiol all fish resembled normal females when sampled at 4 months posthatch. In fish treated with methyltestosterone at all except the lowest dose (25 and 50 μg/l) the gonads resembled neither normal males nor normal females; these gonads were composed largely of connective tissue with only occasional germ cells. Various proportions of male, female and intersex gonads were observed in groups which received androgen or estrogen only in the diet. The implications of the work for salmon culture include the production of sterile fish and increasing the proportion of female salmon in hatchery populations.
The effects of 17β-estradiol and potential xenoestrogens were investigated on Atlantic salmon (Salmo salar). Juvenile salmon fed diets containing 15 and 30 mg 17β-estradiol kg-1 food for 4 weeks after yolk-sac resorption differentiated into 88 and 100% phenotypic females, respectively. The reference group, fed uncontaminated food, consisted of 49% females. A significant overrepresentation of phenotypic females (64%) was recorded in the group fed 1500 mg di-2-ethylhexyl phthalate kg-1 food. Atlantic salmon fed diets contaminated with nonylphenol showed no differences in sex ratios compared with the control group. A significantly increased liver somatic index was detected for groups exposed to diets contaminated with 15 and 30 mg 17β-estradiol, 300 mg nonylphenol or 1500 mg di-2-ethylhexyl phthalate kg-1 food.
Intraperitoneal injections of nonylphenol, di-2-ethylhexyl phthalate, or polychlorinated biphenyls 58, 104, 112 and 188 did not induce synthesis of vitellogenin which is considered to be a hallmark for estrogenic activity in in vivo testing. Therefore, in vivo assays, that is, intraperitoneal injection, can not always predict how endocrine processes in organisms are affected by pollutants.
The zebrafish or zebra danio Danio (=Brachydanio) rerio has recently become a major vertebrate model for the study of developmental biology, neurobiology, and molecular genetics. As a result, most research universities have now invested considerable resources in the construction of large zebrafish facilities. A key element in the design of these facilities is maximizing the efficiency of available space. Here we report on the effects of aquarium chamber volume on the reproduction of zebrafish, with the objective of identifying the minimal volume required for normal egg production. Six adults (two males and four females) were tested in chamber volumes of 500, 400, 300, 200, and 100 mL. Results were compared with those from a control volume of 3.5 L. Eggs were removed from the test chambers after spawning and incubated in petri dishes at 28°C. Total egg production, percent of eggs hatching, and larval length at 96 h postfertilization were used to evaluate breeding success. Compared with the control, egg production was not significantly affected by reduced aquaria volumes of 500, 400, and 300 mL. However, mean egg production from a test volume of 200 mL was only 48% of the control egg production (P < 0.05), and at a test volume of 100 mL, egg production was reduced to 26% of the control value (P < 0.005). Percent egg hatch and 96-h larval length were not affected at any test volume.
The occurrence of hermaphrodite fish in the lagoons of sewage treatment works led us to hypothesize that sewage effluent might contain a substance, or substances, estrogenic to fish. to test this hypothesis, we placed cages containing rainbow trout in the effluent from sewage-treatment works, and one to three weeks later measured the vitellogenin concentration in the plasma of the fish. Vitellogenin is a protein synthesized by the liver of oviparous fish in response to estradiol stimulation; it is then conveyed by the blood to the ovary, where it is sequestered by oocytes to form the yolk. Thus, the presence of vitellogenin in the plasma is indicative of estrogenic stimulation of the liver. an initial study, at a sewage-treatment works, showed that plasma vitellogenin concentrations rose rapidly and very markedly (over 1000-fold in three weeks) when trout were maintained in the effluent. an extensive nationwide survey was then conducted. Results were obtained from fifteen sewage-treatment works distributed throughout England. in all cases, exposure of trout to effluent resulted in a very pronounced increase (500 to 100,000-fold, depending on site) in the plasma vitellogenin concentration. Induction of vitellogenesis was also observed in carp, but to a much lesser extent than in trout.The identity of the estrogenic substance is unknown. It is suggested that the two most likely possibilities are ethynylestradiol, originating from pharmaceutical use, or alkylphenol-ethoxylates (APE), originating from the biodegradation of surfactants and detergents during sewage treatment.Laboratory studies on the potency of ethynylestradiol demonstrated that levels as low as 1 to 10 ng 1 could generate the response shown by the caged fish and that positive responses may arise at 0.1 to 0.5 ng 1. Further work is in progress on the potency of APE.
The roach, Rutilus rutilus, is normally a dioecius species. During an experimental study of reproduction in this species, a condition of apparently functional intersex was discovered. Anatomically the gonad was a testis, exhibiting various normal stages of spermatogenesis. A group of oocytes was found during the microscopical examination of the gonad, interspersed between lobules of normal testicular tissue. A few small oocytes were found to be in the chromatin-nucleolus stage, while the rest were in the perinucleolus stage. In addition, some large oocytes with an average diameter of 40 μm were randomly distributed among the lobules of the testis.
A number of chemicals present in the environment have been shown to mimic or antagonize the actions of steroid hormones, an issue often described as "endocrine disruption/modulation". There is very little evidence, however, to support the hypothesis that exposure to endocrine-disrupting chemicals is a global environmental health problem. In this paper, we demonstrate a high incidence of intersexuality in wild populations of riverine fish (roach; Rutilus rutilus) throughout the United Kingdom. These reproductive disturbances are consistent with exposure to hormonally active substances and are associated with discharges from sewage treatment works that are known to contain estrogenic chemicals. This is the first documented example of a widespread sexual disruption in wild populations of any vertebrate and indicates that reproductive and developmental effects do result from exposure to ambient levels of chemicals present in typical British rivers.
Oral administration of 17β-oestradiol, at a concentration of 20 mg/kg diet, to juvenile rainbow trout and Atlantic salmon, maintained under the natureal photoperiod, for minimum respective periods of 0–30 and 0–21 days following first feeding, induced sex reversal among the males, and resulted in all-female stocks whose gonads were histologically indistinguishable from control females. Shorter periods of oestrogen administration resulted in the production of hermaphrodites in which, characteristically, the testicular components developed more rapidly than ovarian elements. During the period of steroid administration, the fish were more susceptible to adverse conditions and there was some depression of growth. Following the cessation of treatment, the growth rate increased and size differences between treated and control populations, after 150 days, were not significant. Experiments with radioactively labelled oestradiol showed that the steroid enriched diet had an acceptable shelf life and that the half life of oestradiol in rainbow trout was less than 12 h.An alternative method of producing all-female stocks of salmonid fish by mating homogametic individuals in contingent, assuming the males to be heterogametic, on the development of a process for sex reversing the females. Oral administration of 17α methyltestosterone, at a concentration of 3 mg/kg diet, to rainbow trout and salmon for the period 0–90 days after first feeding, induced sex inversion of genetic females and resulted in substantially all-male populations. Treatment of salmon at a dose rate of 30 mg/kg diet resulted in stocks whose gonads had the characteristic filiform appearance of males but in which spermatogonial development had been suppressed.The usefulness of all-female stocks in commercial salmonid rearing operations is discussed.
Xenobiotic pollutants may disrupt reproductive endocrine function by acting at the hypothalamus, pituitary, gonad or liver. Disruption at any of these sites may result in either changes in the rate of gonadal development or in the viability of the gametes. Production of high quality gametes is dependent upon the correct hormonal milieu throughout their development, and any disruption of hormonal balance may result in abnormal sperm or eggs. Such effects may be apparent at levels of pollutant lower than that which causes arrest of gametogenesis or decreased gonadosomatic index. In this paper the use of sperm motility, morphology and fertilising ability in males, and egg numbers, size and hatching success in females as monitors of endocrine disruption will be discussed. Data are presented to show that exposure of adult zebra danios (Danio rerio) to 5 ng l−1 ethynylestradiol leads to arrest in development of the eggs produced at the early blastula stage and induces vitellogenesis in exposed males.
The "Gesetz zum Schutz vor gef~hrlichen Stoffen" (act governing protection against dangerous substances in the FRG), in force since January Ist, 1982, and the 6th Amendment of Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (79/831/EEC) of September 18th, 1979, provide, in testing level 2, for a long-term study on fish which should also take into consideration the effect of substances on reproduction. This is laid down in the act; however, a test method has not yet been elaborated. It seems reasonable to study in a long-term test the effects of substances on growth and also on gamete formation in fish, as these two parameters not only represent important ecological processes but are also sensitive criteria for the toxicological evaluation of substances. Whereas previous studies have focussed on growth in commercial fish species, reproduction was less commonly studied. However, it would be difficult to conduct reproductive studies with most commercial fish species, because most spawn only for a short period of the year and because body size of the adult renders them unsuitable for laboratory use. The fathead minnow, Pimephales promelas RAF., and the flagfish, Jordanella floridae Goode and Bean, were used in these studies predominantly in the USA and Canada (Mount 1973; Smith 1973). Both species are relatively small and can easily be kept in aquaria. In Europe, the zebrafish, Brachydanio rerio HAM.-BUCH., is under discussion as a test species. The fish spawns throughout the year. Earlier published experiments as to the reproduction of this species were reviewed by Laale (1977). Unlike the minnow and the flagfish, the zebrafish does not demonstrate aggressive behaviour in large groups.
The relative potency of several androgens to induce the male phenotype in sexually undifferentiated genotypic female chinook salmon were compared in two separate experiments. The aromatizable and nonaromatizable androgens testosterone (T) and 11-ketotestosterone (11-KT), and the synthetic aromatizable and nonaromatizable androgens 17 alpha-methyltestosterone (MT) and 17 alpha-methyldihydrotestosterone (MDHT) were administered to newly hatched alevins in a single 2-hr immersion treatment at doses ranging from 3.2 micrograms/liter to 10 mg/liter. The influence of these treatments on sex differentiation was evaluated by the histological examination of the resulting gonads 6 and 11 months later. In the control group, which was not exposed to exogenous steroids, no males or intersex fish were observed. In contrast, essentially 100% masculinization occurred in groups exposed to MDHT at dosages of 400 micrograms/liter and higher. Treatment with the aromatizable androgen MT resulted in a dose-dependent masculinization, with the production of 100% males at 400 micrograms/liter. However, higher doses resulted in fewer males. 11-KT and T were less potent than the synthetic androgens. The number of males produced after treatment with 11-KT followed a dose-dependent pattern while T showed virtually no masculinizing effect in inducing male phenotype in these studies. The resultant AD50 dosage levels (dosage at which 50% of the genotypic females were sex-reversed into phenotypic males) after a single 2-hr immersion treatment were: 30, 60, and 500 micrograms/liter for MDHT, MT, and 11-KT, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
This review describes the research that has been carried out into estrogenic effects occurring in aquatic environments, both freshwater and marine, and the substances found to be responsible. In summary, estrogenic (and probably some anti-androgenic) activity has mainly been detected in a variety of treated sewage and other effluents, but also as a result of certain chemical spills and deliberate applications. This activity has resulted in a number of effects in vertebrate wildlife that can best be described as feminization, although the severity of these effects ranges from biomarkers of exposure such as vitellogenin induction in males through to morphological changes in sex organs and complete sex reversal. The implications of these changes for the future of aquatic wildlife populations have not yet been thoroughly explored. It is unlikely that all the causative substances have yet been discovered, but those which have been positively identified include natural and synthetic estrogenic hormones, natural plant sterols, synthetic alkylphenols, and certain organochlorine substances. The review concludes that there is now a need to investigate the consequences for wildlife populations of exposure to these materials, by means of a variety of field experiments and investigations.
Definitive data on reproductive impairment of chronically exposed populations may be required to assess the appropriateness of the existing test methods for hazard identification and prioritization of endocrine modulators. Multigeneration toxicity testing protocols for wildlife receptors are lacking. To help address this gap we describe a multigeneration fish assay using the freshwater fish, Japanese medake (Oryzias latipes). This test species has been used for the evaluation of carcinogenic, teratogenic and reproductive effects and is sensitive to estrogen exposure producing ovo-testis, altered biochemical parameters and phenotypic characteristics. Due to the short life cycle, a multigeneration test with medaka can be conducted in 1 year. Endpoints evaluated include: survival, growth, sex ratio, fecundity, embryonic lesion occurrence, embryonic stage development, gonadal and hepatic somatic indices, histopathology and biochemical parameters. As new endpoints are developed they can be incorporated into the protocol. Results of a positive control (17 beta-estradiol) study are presented to give an indication of the baseline associated with various test endpoints and to highlight the importance of nutrition in the experimental design. 17 beta-Estradiol treatment induced vitellogenin production in male and female medaka, feminized males, and disrupted egg production. The proposed protocol provides researchers with an effective multigeneration fish test that can be used to examine potential effects of stressors at the population, individual, cellular and subcellular level.
Further studies on the embryoni c development of the zebrafish
- K Hisaoka
- C Firlit
Hisaoka, K., Firlit, C., 1960. Further studies on the embryoni c development of the zebrafish, Brachydanio rerio (Hamilton-Buchanan). J. Morphol 107, 205-225.
The normal development stages of the zebrafish
- K Hisaoka
- H Battle
Hisaoka, K., Battle, H., 1958. The normal development stages of the zebrafish, Brachydanio rerio (Hamilton-Buchanan).
Sex control and sex reversal
- S T H Chan
- W S B Yeung
Chan, S.T.H., Yeung, W.S.B., 1983. Sex control and sex reversal.
Development and reproduction of roach (Rutilus rutilus) in the Szczecin firth
- A Peczalska
Peczalska, A., 1968. Development and reproduction of roach (Rutilus rutilus) in the Szczecin firth. Pol. Arch. Hydrobiol. 15, 103-120.
Summary of the workshop 'Reproduction disturbances in fish
- L Norrgren
- Be Bengtson
- H Börjeson
Norrgren, L., Bengtson, BE., Börjeson, H., 1994. Summary of the workshop 'Reproduction disturbances in fish'. In: L.
Report from the Uppsala worksho p on reproduction disturbances in fish
- Norrgren
Norrgren, (Ed.). Report from the Uppsala worksho p on reproduction disturbances in fish, 20-22 October 1993. Swedish Environmental Protection agency. Report 4346, Solna.
Assessment of estrogenic effects in the zebrafish, Danio rerio. 6 t h International Symposiu m on Reproductive Physiology of Fish
- M Fenske
- G Maack
- U Ensenbach
- H Segner
Fenske, M., Maack, G., Ensenbach, U., Segner, H., 1999. Assessment of estrogenic effects in the zebrafish, Danio rerio. 6 t h International Symposiu m on Reproductive Physiology of Fish, Bergen, Norway, July 4-9, 1999.