The expression of CYP1A, vitellogenin and zona radiata proteins in Atlantic salmon (Salmo salar) after oral dosing with two commercial PBDE flame retardant mixtures: absence of short-term responses

The short-term effects of the commercial PBDE flame retardant mixtures Penta-BDE and Octa-BDE on the expression of cytochrome P450 1A (CYP1A), vitellogenin (Vtg) and zona radiata proteins (Zrp) were investigated in juvenile salmon (Salmo salar). For this purpose, groups of fish were dosed twice (oral intake at days 1 and 4) with 10 and 50 mg/kg body weight of both commercial mixtures. The fishes were sacrificed at day 7 (n=5 for each group) and 14 (n=6 for each group), and blood, liver, fillet, and brain were collected. Blanks and positive controls were also part of the experiment. The expressions of Vtg, Zrp, and CYP1A were measured with several techniques (EROD, ELISA, Western, Northern and Slot Blot). The values in the groups of fish treated with Penta-BDE or Octa-BDE did not significantly differ from the reference group for any of the parameters tested. In contrast, the positive control groups treated with estradiol-17β for Vtg and Zrp expression, and β-naphthoflavone for CYP1A expression did show a significant response, indicating the potential sensitivity of the fishes for the parameters measured. Since the results of the chemical analyses showed concentrations of a number of PBDE congeners in liver, fillet, and brain that were about three orders of magnitude above those of fish from the North Sea, it is concluded that the short-term toxicity of both commercial PBDE mixtures for these endpoints was low.     

o,p′-DDT induction of vitellogenesis and its inhibition by tamoxifen in Nile tilapia (Oreochromis niloticus)

In order to investigate the mechanism by which o,p′-DDT disrupts endocrine functioning of Nile tilapia in vivo, the estrogenicity of o,p′-DDT was investigated in conjunction with 17β-estradiol (E₂) and tamoxifen. Mature, male tilapia were treated intraperitoneally with o,p′-DDT (60 mg/kg, one dose) or E₂ (5 mg/kg, four doses) in the presence or absence of tamoxifen (5 mg/kg, six doses) for 12 days and then plasma vitellogenin (Vtg) (measured as alkaline-labile phosphorous), E₂, and testosterone (T) were measured. Vtg levels were increased dramatically by E₂ (1744±171 μg/ml) and moderately by o,p′-DDT (82±15 μg/ml) compared with controls (23±3.5 μg/ml). Tamoxifen alone had no effect on Vtg production, but inhibited both E₂ and o,p′-DDT stimulated vitellogenesis. T levels were reduced with E₂ administration (1688±383 pg/ml) and declined further with the combined treatment of E₂ and tamoxifen (281±70 pg/ml), compared with controls (6558±1438 pg/ml). Tamoxifen or o,p′-DDT alone did not affect T levels, but their combined treatment did (2069±647 pg/ml). The results of this study suggest that o,p′-DDT is weakly estrogenic in male tilapia, and that this activity may be mediated through the estrogen receptor.     

Estrogenic activity in sediments contaminated by nonylphenol in Tokyo Bay (Japan) evaluated by vitellogenin induction in male mummichogs (Fundulus heteroclitus)

Estrogenic activity was determined in sediments collected from Tokyo Bay. Sampling was performed at five stations including the site near the sewage treatment plant. The most estrogenic sediment collected near the sewage treatment plant was fractionated into ten fractions using normal-phase high-performance liquid chromatography. Chemical analysis was carried out for each fraction and nonylphenol (NP, 20,700 ng g⁻¹ dry wt) was detected at a higher concentration than estron (2.39 ng g⁻¹ dry wt) and 17β-estradiol (<0.7 ng g⁻¹ dry wt). Furthermore, each fraction was administered to male mummichogs (Fundulus heteroclitus), and vitellogenin (Vtg) was measured after two weeks. The induction of Vtg was observed; this estrogenic potency could be attributed to the NP content in this fraction. This is the first report to suggest that the high NP concentration in the sediments from Tokyo Bay has the potential to induce Vtg in wild fish.     

A survey of indicators for reproductive endocrine disruption in Fundulus heteroclitus (killifish) at selected sites in the Chesapeake Bay

Plasma vitellogenin and related parameters in the killifish Fundulus heteroclitus were measured at selected sites in the Chesapeake Bay. In males, vitellogenin was above the detection limit 14% of the time, and detections did not differ between sites or seasons. Few differences in plasma vitellogenin levels were found between sites during fall in either male or female F. heteroclitus, the time of natural gonadal regression for this species. There was some variation in the ratio of male to female F. heteroclitus, but was not consistent at most sites. Significant negative correlations were found between reported sediment polycyclic aromatic hydrocarbons (PAHs) and GSI, and PAHs and plasma vitellogenin in females in both Spring 1999 and Spring 2000. Gonadal anomalies in F. heteroclitus included slight reductions in certain tissue types. Overall, reproductive endocrine disruption in the killifish F. heteroclitus at the sites sampled in the Chesapeake Bay appeared somewhat minimal.     

市政污水厂尾水对文蛤卵黄蛋白原的诱导效应研究

本研究目的是检验市政污水厂尾水排放对海洋双壳类动物文蛤的内分泌干扰效应,判断其血浆中卵黄蛋白原(Vtg)水平作为尾水受纳海域雌激素效应生物标志物可行性。将文蛤在不同浓度的尾水-海水混合液(0%、1%、5%、10%、20%、40%)中持续暴露培养20d,定期测定雄性文蛤血浆中Vtg含量,分析暴露期间Vtg水平随尾水体积比(EVR)和培养时间的变化趋势。结果表明,暴露培养10d后,尾水体积比40%的处理组中雄性文蛤的血浆Vtg含量明显上升(P0.05),随着培养时间延长到15和20d,在较低的EVR(10%、20%)下也能监测到尾水对文蛤Vtg的明显诱导,表明污水处理厂尾水中存在一定数量的内分泌活性物质,对雄性文蛤具有显著类雌激素效应;Vtg最大值出现在EVR20%的尾水暴露15d以及EVR40%的尾水暴露20d,分别比对照组增加45.86%和47.40%。如果采用笼养文蛤监测尾水受纳海域的类雌激素效应,适宜的暴露时间为20d,此时采集的雄性文蛤Vtg可指示较大范围尾水的影响。     

Are steroids really the cause for fish feminization? A mini-review of in vitro and in vivo guided TIEs

Feminization of fish has been reported throughout the world in freshwater and marine systems. While the population impacts are conflictive, enough negative effects warrant additional research into causation. In order to ascertain the identities of specific feminizing agents, variants of toxicity identification evaluations (TIEs) have been employed. The majority of these evaluations have utilized in vitro estrogen receptor-based cell-lines to identify chromatographic fractions that possess biological activity from predominately wastewater derived from municipal treatment facilities and have concluded that synthetic and natural estrogens are the primary cause for feminization of fish. This paper will focus on three aquatic systems impacted by wastewater originating from purely domestic, and industrial/domestic secondary treatment systems. Wastewater and sediment extracts were evaluated by in vitro and in vivo biological responses in a TIE fractionation design. While in vitro responses tended to mirror in vivo responses in purely domestic wastewater systems, in vitro responses tended to severely underestimate in vivo estrogenic activity when normalized to estradiol equivalents in more complex systems. TIE fractionation schemes using in vivo biological responses failed to indicate any relationship to steroids in either wastewater or sediment extractions. These data consistently support the view that mechanisms other than direct ER binding and activation by toxicants may be important in the feminization of fish particularly residing in habitats that receive complex wastewater or agricultural effluents.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0–0.2 mg NP l⁻¹), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC₅₀ value was previously determined. Then, in sublethally exposed (0–1.0 mg NP l⁻¹) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 μg g⁻¹ d.w. in mussels, and from 3.6 to 37 μg g⁻¹ d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.     

Endocrine Disrupters in the Aquatic Environment: An Overview

Although a relatively new area of environmental research, the field of endocrine disruption has grown very rapidly, and currently many hundreds, perhaps even a few thousand, papers are published annually on the many different aspects covered by the field. As far as endocrine disruption in wildlife is concerned, most attention has been focused on aquatic organisms, for two reasons. Firstly, the aquatic environment receives most of the pollutants intentionally released into the environment, through effluents from wastewater treatment plants, and secondly because many of the best documented examples of endocrine disruption in wildlife are of partially or completely aquatic species. These two reasons are probably not unconnected, of course. Hence, aquatic organisms can receive continuous exposure to endocrine‐disrupting chemicals throughout their lives, albeit usually to low concentrations of these chemicals. Analysis of effluents has identified many of the endocrine‐disrupting chemicals present, and shown that these are both natural and man‐made, and vary greatly in potency. Most attention has been directed to identifying the main estrogenic chemicals, because many of the effects reported in wildlife appear to be a consequence of ‘feminization’ of males. However, chemical analysis of effluents has also demonstrated that chemicals with other types of endocrine activity are present, such as androgens, anti‐androgens, progestagens, etc. The effects (if any, of course) of such chemicals on aquatic organisms are unknown, and largely uninvestigated, presently. Much of the biological research has centred on the effects of estrogenic chemicals, especially to fish. These effects, such as elevated vitellogenin concentrations and intersexuality, have to date been studied almost exclusively at the level of the individual, and hence whether endocrine‐disrupting chemicals cause population‐level consequences is largely unknown (the undeniable effects of TBT on molluscs, leading to local extinctions, being the exception). It is my opinion that rather too much of the recent research has not advanced our understanding of endocrine disruption a great deal, and we are probably not much further forward now than we were five years or so ago. It is surely time to tackle some of the outstanding, unresolved issues, such as the impact of endocrine disruption at the population level, and the issue of how organisms respond when exposed to complex mixtures of endocrine active chemicals. Such research will not be easy, and will require multidisciplinary teams, including people with expertise in areas not yet involved in the field of endocrine disruption, such as mathematical modellers. However, until such research is done, it will not be possible to decide how important an issue endocrine disruption is to wildlife, and how that importance compares to the other factors adversely affecting wildlife, such as habitat loss, climate change, and the introduction of exotic species and novel diseases.     

Estrogenic and CYP1A response of mummichogs and sunshine bass to sewage effluent

Recent studies demonstrating feminization of effluent-exposed wild-caught male fish in the UK have prompted much research regarding the estrogenic activity of effluent from municipal sewage treatment plants (MSTPs). To investigate the estrogenicity and cytochrome P450 1A (CYP1A) induction potency of MSTP effluent, two species of fish, adult male mummichogs, Fundulus heteroclitus, and juvenile sunshine bass, Morone saxatilis x Morone chrysops, were exposed to un-chlorinated effluent (75% effluent, 25% seawater) from a large MSTP in Yonkers, NY, USA. After a 21-day static-daily (75%) renewal exposure, significant elevations over controls were observed in levels of vitellogenin (VtG) in plasma (1730%) and liver (131%) in effluent-exposed sunshine bass. In contrast, hepatic VtG was not elevated in mummichogs; plasma VtG was not measured in this species. Effluent exposure elevated hepatic CYP1A protein (140-145%) and ethoxyresorufin-O-deethylase (EROD) activity (408-598%) in both species. These findings suggest ontogenetic and/or species differences in response to estrogenic compounds in MSTP effluent. Furthermore, the elevation of CYP1A in response to sewage effluent exposure indicates the presence of additional compounds that may alter xenobiotic and/or steroid biotransformation in fish.     

Vitellogenin as biomarker for estrogenicity in flounder Platichthys flesus in the field and exposed to 17α-ethinylestradiol via food and water in the laboratory

The ability of 17α-ethinylestradiol (EE2) to elevate vitellogenin levels were investigated in male flounder Platichthys flesus and vitellogenin concentrations in flounders from the Danish coastal environment were determined. Male flounders were exposed to 17α-ethinylestradiol (EE2) via food or water. Average vitellogenin concentrations in the control fish ranged between 25 and 100 ng mL⁻¹. Exposure to 5.1, 8.1 and 16.8 ng EE2 L⁻¹ in water and 500 and 5000 ng EE2 kg⁻¹ body weight (bw) every second day in the food increased the plasma vitellogenin concentration in a concentration and time dependent manner, whereas exposure to 2.7 ng EE2 L⁻¹ in water for 21 d and 5 and 50 ng EE2 kg⁻¹ bw for 12 days in the food did not. EE2 could be detected in liver and testes (but not in muscle) after exposure to 8.1 and 16.8 ng EE2 L⁻¹ in the water and 5000 ng EE2 kg⁻¹ bw in the food; the highest concentration was 6 ng g⁻¹ wet weight in liver. The majority of the male flounders collected from nine coastal Danish sites from 1999 to 2004 had vitellogenin concentrations below 100 ng mL⁻¹, and only at two sites moderate estrogenic inputs were indicated.