Plasma Vitellogenin Concentrations Research Articles

Binding Affinities of Hepatic Nuclear Estrogen Receptors for Genistein in Channel Catfish

AbstractSoybean meal, the major protein source used in commercial feeds for channel catfish Ictalurus punctatus, contains natural phytoestrogens known as isoflavones, many of which have been found to be endocrine disruptors. This study investigated whether genistein (an isoflavone) in a soybean‐based diet would increase plasma vitellogenin (Vtg) concentrations and whether it would compete with estradiol (E2) for binding sites on adult channel catfish hepatic estrogen receptors (ERs). Three‐year‐old channel catfish males and females were fed a casein‐based diet (control) or a soybean‐based diet. The genistein concentrations were 93.2 ng/g and 415,800 ng/g in the control and soybean diets, respectively. The binding specificity of the ERs was assessed by binding‐affinity studies. In order of decreasing binding affinity, ethinylestradiol > E2 > genistein > testosterone, demonstrating that the hepatic ERs in channel catfish bind estrogens and genistein more than androgens. No significant differences in affinity (Kd ) were observed for hepatic ERs in male or female catfish fed the soybean diet compared with those fed the control diet. Significant increases in binding capacity (B max) for males fed the soybean diet (158 ± 15 fmol/mg protein) were evident compared with males fed the control diet (73 ± 4 fmol/mg protein). Genistein competed with [3H]E2 for hepatic ERs in both the male and female channel catfish. The relative binding affinities in male ERs were higher (2.72) than those in female ERs (1.17) for genistein. Because diets of farm‐raised channel catfish are routinely composed of high quantities of soybean meal, the findings that genistein stimulates Vtg production in male channel catfish and binds to the ERs in both male and female channel catfish suggest that dietary genistein could alter production traits. However, further research to elucidate the effects of genistein on catfish reproduction is necessary to determine the impact of this phytoestrogen on catfish physiology.

Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme‐linked immunoassay in the fathead minnow (Pimephales promelas)

The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spectrometry (LC/ESI-MS/MS), and two commercial enzyme-linked immunoassay (ELISA) kits using either anti-carp or anti-FHM antibodies. The influence on plasma VTG measurements of using the protease-inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze-thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze-thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI-MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM.

Predicted exposures to steroid estrogens in U.K. rivers correlate with widespread sexual disruption in wild fish populations.

Steroidal estrogens, originating principally from human excretion, are likely to play a major role in causing widespread endocrine disruption in wild populations of the roach (Rutilus rutilus), a common cyprinid fish, in rivers contaminated by treated sewage effluents. Given the extent of this problem, risk assessment models are needed to predict the location and severity of endocrine disruption in river catchments and to identify areas where regulation of sewage discharges to remove these contaminants is necessary. In this study we attempted to correlate the extent of endocrine disruption in roach in British rivers, with their predicted exposure to steroid estrogens derived from the human population. The predictions of steroid estrogen exposure at each river site were determined by combining the modeled concentrations of the individual steroid estrogens [17β -estradiol (E2), estrone (E1), and 17α -ethinylestradiol (EE2)] in each sewage effluent with their predicted dilution in the immediate receiving water. This model was applied to 45 sites on 39 rivers throughout the United Kingdom. Each site studied was then categorized as either high, medium, or low “risk” on the basis of the assumed additive potency of the three steroid estrogens calculated from data derived from published studies in various cyprinid fish species. We sampled 1,438 wild roach from the predicted high-, medium-, and low-risk river sites and examined them for evidence and severity of endocrine disruption. Both the incidence and the severity of intersex in wild roach were significantly correlated with the predicted concentrations of the natural estrogens (E1 and E2) and the synthetic contraceptive pill estrogen (EE2) present. Predicted steroid estrogen exposure was, however, less well correlated with the plasma vitellogenin concentration measured in the same fish. Moreover, we found no correlation between any of the end points measured in the roach and the proportion of industrial effluents entering the rivers we studied. Overall, our results provide further and substantive evidence to support the hypothesis that steroidal estrogens play a major role in causing intersex in wild freshwater fish in rivers in the United Kingdom and clearly show that the location and severity of these endocrine-disrupting effects can be predicted.

Characterization of Environmental Estrogens in River Water Using a Three Pronged Approach: Active and Passive Water Sampling and the Analysis of Accumulated Estrogens in the Bile of Caged Fish

Estrogenicity of river water is highly variable and it is difficult to obtain an average measure of the estrogenicity. Consequently it is difficult to tie the estrogenic effects observed in fish to their level of exposure to estrogens. To get a better handle on average estrogenic exposure we tested a recently developed passive sampling system (polar organic chemical integrative sampler, POCIS). In addition, we investigated the bioaccumulation of estrogens in caged brown trout and measured plasma vitellogenin in males as a bioindicator of estrogenic effects. We developed a mini-caging method to suit the hydrological conditions in small rivers and to improve upon the often poor survival of salmonids in caging trials. POCISs were positioned upstream and downstream of 5 sewage treatment works' discharges and left on site for 3 weeks (as were the caged fish), during which period 3 water grab samples were taken at each site. Concentrations of estrogens were determined using a yeast-based reporter gene assay and chemical analysis. Results from grab sampling, passive sampling, and bioaccumulation were correlated; however, plasma vitellogenin concentrations were elevated at only 1 of 5 sites. POCISs provide an integrated and biologically meaningful measure of estrogenicity in thatthey accumulate estrogens in a pattern similar to that of brown trout. Mini-caging appears a significant methodological advance; no fish were lost, moreover, all fish survived in excellent health.

Characterization of the estrogenicity of Swiss midland rivers using a recombinant yeast bioassay and plasma vitellogenin concentrations in feral male brown trout

In our study, we aim to characterize the estrogenicity of 18 independent rivers that receive effluent from sewage treatment works. During the winter and summer of 2003, we collected multiple water samples and measured environmental estrogens with an in vitro yeast-based reporter gene assay; estrogenicity was expressed as ng 17beta-estradiol equivalents (EEQ) per L of water. Estradiol equivalents values in winter ranged from 0.3 to 2.0 ng/L and, in summer, from 0.4 to 7.0 ng/L. Winter and summer EEQ values were not correlated with each other or with the dilution factor of the effluent in the river. Variation in EEQ values was large and correlated from winter to summer. Part of this variation in estrogenicity is explained by water flow rates; variation is larger at reduced flow rates. We measured plasma vitellogenin concentrations in immature male brown trout. At five sites, vitellogenin concentrations exceeded 1 microg/ml; however, at the majority of the sites, plasma vitellogenin concentrations were below 0.5 microg/ml. Our data indicate that the exposure of brown trout to environmental estrogens in Swiss midland rivers is low. However, some sites show reoccurring higher EEQ values and, at some sites, plasma vitellogenin concentrations in male fish clearly are elevated.

Environmental contaminants and biomarker responses in fish from the Rio Grande and its U.S. tributaries: Spatial and temporal trends

We collected, examined, and analyzed 368 fish of seven species from 10 sites on rivers of the Rio Grande Basin (RGB) during late 1997 and early 1998 to document temporal and geographic trends in the concentrations of accumulative contaminants and to assess contaminant effects on the fish. Sites were located on the mainstem of the Rio Grande and on the Arroyo Colorado and Pecos River in Texas (TX), New Mexico (NM), and Colorado. Common carp ( Cyprinus carpio) and largemouth bass ( Micropterus salmoides) were the targeted species. Fish were examined in the field for internal and external visible gross lesions, selected organs were weighed to compute ponderal and organosomatic indices, and samples of tissues and fluids were obtained and preserved for analysis of fish health and reproductive biomarkers. Whole fish from each station were composited by species and gender and analyzed for organochlorine chemical residues and elemental contaminants using instrumental methods, and for 2,3,7,8-tetrachloro dibenzo- p-dioxin-like activity (TCDD-EQ) using the H4IIE rat hepatoma cell bioassay. Overall, fish from lower RGB stations contained greater concentrations of organochlorine pesticide residues and appeared to be less healthy than those from sites in the central and upper parts of the basin, as indicated by a general gradient of residue concentrations and biomarker responses. A minimal number of altered biomarkers and few or no elevated contaminant concentrations were noted in fish from the upper RGB. The exception was elevated concentrations [up to 0.46 μg/g wet-weight (ww)] of total mercury (Hg) in predatory species from the Rio Grande at Elephant Butte Reservoir, NM, a condition documented in previous studies. Arsenic (As) and selenium (Se) concentrations were greatest in fish from sites in the central RGB; Se concentrations in fish from the Pecos River at Red Bluff Lake, TX and from the Rio Grande at Langtry, TX and Amistad International Reservoir, TX exceeded published fish and wildlife toxicity thresholds. In the lower RGB, residues of p,p′-DDT metabolites (≤1.69 μg/g ww), chlordane-related compounds (≤0.21 μg/g ww), dieldrin (≤0.0.05 μg/g ww), and toxaphene (≤2.4 μg/g ww) were detected in fish from most sites; maximum concentrations were in channel catfish ( Ictalurus punctatus) from the Arroyo Colorado at Harlingen, TX. Concentrations of one or more residues exceeded toxicity thresholds for fish and wildlife in fish from this site and from the Rio Grande at Mission, TX and Brownsville, TX; however, concentrations were lower than those reported by previous studies. In addition, the proportional concentrations of p,p′-DDT at all sites were low, indicating weathered DDT rather than the influx of new material. Concentrations of total PCBs (<0.05 μg/g ww) and TCDD-EQ (≤6 pg/g ww) were comparatively low in all samples. Hepatic ethoxyresorufin O-deethylase (EROD) activity in some fish was elevated relative to reference rates at most sites, but was generally lower than previously reported activity in fish from heavily contaminated locations. The comparatively low PCB and TCDD-EQ concentrations together with elevated EROD activity may reflect exposure to polycyclic aromatic hydrocarbons. Reproductive biomarkers were consistent with chronic contaminant exposure at lower RGB sites; comparatively large percentages of intersex male largemouth bass, relatively low gonadosomatic indices, and elevated plasma vitellogenin concentrations in male fish were noted at three of the four stations. Large percentages of atretic eggs were also observed in the ovaries of female common carp from the Rio Grande at Brownsville, TX. Although many of the conditions noted may have other causes in addition to contaminant exposure, the biomarker results for the lower RGB sites are consistent with subtle responses of fish to contaminants, an interpretation supported by the chemical data of this and other investigations.

Effects of 2,2′4,4′5,5′-hexachlorobiphenyl (PCB 153) on plasma sex steroids and vitellogenin in rockfish ( Sebastes schlegeli)

We examined the estrogenic effect of 2,2′4,4′5,5′-hexachlorobiphenyl (PCB 153) on the rockfish, Sebastes schlegeli. We measured levels of plasma estradiol-17β (E 2) and testosterone (T) by radioimmunoassay (RIA). Plasma concentrations of T and 17α-hydroxyprogesterone in female and male fish injected with PCB 153 using two dosages (0.16 mg/kg body weight. and 0.57 mg/kg) did not differ significantly between sexes or from sham-injected controls of the same sex. Plasma concentrations of E 2 in females injected with PCB 153 (both levels) increased at 12 and 24 h. Concentrations of plasma vitellogenin (VTG) in females increased 72 h after injection with PCB 153 and reached 0.38 and 1.25 mg/mL, respectively. No VTG was detected in males injected with the same dosage. These results suggest that PCB 153 may lead to the production VTG in female rockfish through a synergistic effect with E 2, resulting in indirect disruption of the aromatization process.

An integrated assessment of estrogenic contamination and biological effects in the aquatic environment of The Netherlands

An extensive study was carried out in the Netherlands on the occurrence of a number of estrogenic compounds in surface water, sediment, biota, wastewater, rainwater and on the associated effects in fish. Compounds investigated included natural and synthetic hormones, phthalates, alkylphenol(ethoxylate)s and bisphenol-A. The results showed that almost all selected (xeno-)estrogens were present at low concentrations in the aquatic environment. Locally, they were found at higher levels. Hormones and nonylphenol(ethoxylate)s were present in concentrations that are reportedly high enough to cause estrogenic effects in fish. Field surveys did not disclose significant estrogenic effects in male flounder ( Platichthys flesus) in the open sea and in Dutch estuaries. Minor to moderate estrogenic effects were observed in bream ( Abramis brama) in major inland surface waters such as lowland rivers and a harbor area. The prevalence of feminizing effects in male fish is largest in small regional surface waters that are strongly influenced by sources of potential hormone-disrupting compounds. High concentrations of plasma vitellogenin and an increased prevalence of ovotestes occurred in wild male bream in a small river receiving a considerable load of effluent from a large sewage treatment plant. After employing in vitro and in vivo bioassays, both in situ and in the laboratory, we conclude that in this case hormones (especially 17α-ethynylestradiol) and possibly also nonylphenol(ethoxylate)s are primarily responsible for these effects.

Reproductive Responses of Common Carp (Cyprinus carpio) Exposed in Cages to Influent of the Las Vegas Wash in Lake Mead, Nevada, from Late Winter to Early Spring

The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent, nonpotable shallow groundwater seepage, and runoff from the urbanized Las Vegas Valley to Las Vegas Bay (LX) of Lake Mead. To investigate the potential for contaminants in LW influent to produce effects indicative of endocrine disruption in vivo, adult male and female common carp (Cyprinus carpio) were exposed in cages for 42-48 d at four sites in Lake Mead: LW, LX, and two reference locations in the lake. End points examined included gonadosomatic index; gonad histology; concentrations of plasma vitellogenin (VTG) and plasma sex steroids (17beta-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT)); plasma estrogen:androgen ratios (E2:T, E2:11-KT), in vitro production of T by gonad tissue, and hepatopancreas ethoxyresorufin O-deethylase activity. Few differences among fish caged at different sites were potentially attributable to exposure to contaminants

Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout ( Zoarces viviparus)

Estrogenic chemicals such as alkylphenols (APs) have been shown to disrupt the reproductive system of male fish. In the present study, the effects of the estrogenic chemical octylphenol (100 μg g −1) and 17β-estradiol on the semen production and the biochemical composition of the seminal fluid of the viviparous eelpout ( Zoarces viviparus) were investigated at the time of spawning. After 10 days of octylphenol or estrogen treatment, vitellogenin (Vtg) synthesis was induced as indicated by increased plasma vitellogenin concentration. In accordance with the increased vitellogenin concentration, hepatosomatic index (HSI), total protein concentration, and total calcium concentration were also increased, and free amino acids concentration was decreased in blood plasma. Octylphenol treatment caused a decrease in the gonadosomatic index (GSI) and the milt volume and an increase in the spermatocrit. The histological examination revealed that octylphenol affected the normal lobular structure, including the Sertoli cells. In the majority of the octylphenol-treated fish, trapped sperm cells were observed in parts of the seminiferous lobules and the sperm ducts. The biochemical composition of the seminal fluid was also affected by the octylphenol or estrogen. The seminal plasma concentrations of magnesium, calcium, and total protein were elevated, and the concentration of free amino acids was reduced in the treated fish. This study indicates that octylphenol inhibits the seminal fluid production and changes its biochemical composition in eelpouts.

Successful detection of (anti-)androgenic and aromatase inhibitors in pre-spawning adult fathead minnows ( Pimephales promelas) using easily measured endpoints of sexual development

Screening assays have been successfully developed for the detection of (anti-)oestrogenic substances in several fish species, including the fathead minnow ( Pimephales promelas). Previous work suggested that pre-spawning adult fathead minnows might be an appropriate life-stage for developing a screen to detect endocrine active substances (EASs). Pre-spawning adult fathead minnows, in which their phenotypic sex could be determined, were exposed in flow-through systems to three reference substances for 21 days, at 25 °C. Male and female fish, held in separate tanks, were exposed to dihydrotestosterone (DHT, androgen), flutamide (anti-androgen) and fadrozole (aromatase inhibitor). Nominal (mean measured) concentrations for DHT were 10 (6.0), 32 (6.1) and 100 (8.6) μg l −1, for flutamide, 100 (95.3), 320 (320.4) and 1000 (938.6) μg l −1 and for fadrozole, 25 (24.8), 50 (51.7) and 100 (95.5) μg l −1. After 14 and 21 days exposure, fish were evaluated for growth, secondary sexual characteristics (SSCs, number and prominence of nuptial tubercles), gonadosomatic index (GSI) and plasma vitellogenin (VTG) concentrations. Development of nuptial tubercles was sensitive to both DHT and flutamide exposure. Exposure to DHT significantly increased the number of nuptial tubercles (male characteristic) in both males (more abundant) and females, after 14 days. Flutamide (938.6 μg l −1, day 21) significantly reduced nuptial tubercle number in male fish. Fadrozole significantly inhibited ovarian growth (lower GSI) and significantly induced testis growth (51.7 and 95.5 μg l −1), after 21 days. Plasma VTG concentrations were significantly elevated in male fish (6.1 and 8.6 μg l −1), but inhibited in female fish (6.0 μg l −1), exposed to DHT. Flutamide had no effect on plasma VTG in male fish, but significantly induced VTG in female fish, after 21 days. Fadrozole significantly inhibited VTG in females and induced VTG synthesis in males, at day 21. These results show that SSCs, GSI and plasma VTG concentrations can be used in pre-spawning adult fathead minnows to screen for a range of classes of EASs. This work complements other published studies in supporting the current OECD effort towards validating a 21 days non-spawning fish screening assay for assessing (anti-)oestrogens, aromatase inhibitors and (anti-)androgens.

Effects of atrazine on fathead minnow in a short-term reproduction assay.

Atrazine is the most extensively used herbicide in the United States. Part-per-million concentrations of atrazine have been reported in agricultural runoff. It is detectable in surface waters and precipitation throughout the year, and it has been found in groundwater sources of drinking water. Recent studies indicate that atrazine may be a potent endocrine-disrupting compound in frogs exposed to part-per-billion (microg/L) concentrations. For these reasons, the effects of atrazine (5 and 50 microg/L) on several endpoints related to reproductive fitness were examined in fathead minnows (Pimephales promelas) in a 21-d static exposure. Estradiol (0.5 microg/L) was included as a positive-control treatment. Endpoints examined in adult fish during and after the exposures included survival, egg production, number of spawns, eggs/spawn, relative gonad weight, gonad histology, number of nuptial tubercles, and plasma vitellogenin concentration. Eggs produced during the exposures were hatched and reared in control water. The percentages of embryos fertilized and hatched as well as larval survival were evaluated. Decreasing trends were observed in relative testis weight, testis maturity, and percentage embryo fertilization. These trends suggest that further investigation is warranted, but the differences in these and other endpoints were not statistically significant in the atrazine-exposed fish. Nearly all endpoints concerning fish exposed to estradiol were significantly different from atrazine-exposed fish and control fish. These results suggest that atrazine did not have strong estrogenic effects in adult fathead minnows and did not cause overt reproductive toxicity at environmentally relevant concentrations.

Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish ( Carassius auratus)

Sexually mature goldfish ( Carassius auratus) of both sexes were exposed to two doses (100 and 1000 μg/l) of the widely used herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino- s-triazine) for a period of 21 days and effects on the concentrations of gonad and plasma sex steroids (testosterone (T), 17β-estradiol (E2) and 11-ketotestosterone (11-KT)), plasma vitellogenin (VTG) and gonad histo-morphology assessed. Atrazine did not show any obvious estrogenic effect in males, as determined by a lack of vitellogenin induction. There were, however, effects of atrazine on plasma androgen concentrations (androgen dynamics) and tissue (plasma and gonad) estrogen concentrations in male goldfish; exposure to 1000 μg/l atrazine induced suppression in both testosterone and 11-ketotestosterone, and resulted in elevated 17β-estradiol, after 21 day of exposure. Further, these suppressive effects on plasma androgens and the induction in estrogen were dose- and time-related. The highest atrazine exposure dose induced structural disruption in the testis and both 100 and 1000 μg/l induced elevated levels of atresia in ovaries.

Development of Plasma Vitellogenin Assay for Estrogenic Effects of Endocrine-Disrupting Chemicals Using Ovariectomized Goldfish (Carassius auratus)

Plasma vitellogenin (VTG) assay was developed using ovariectomized goldfish (Carassius auratus) for determining the estrogenic effects of endocrine-disrupting chemicals. In a laboratory study, we assessed the estrogenic activity of commercial fish diets by using a diet for ornamental carp (CD) and a casein-based formulated fish diet (FD), which was shown to not contain soybean or fish meal in a previous study. In ovariectomized fish, plasma VTG concentrations were significantly higher in the CD-fed group than in the FD-fed group. These results indicate that the estrogen activity of CD may be high enough to cause induction of plasma VTG in ovariectomized goldfish as previously observed in male goldfish. Moreover, the effect of estrogen on plasma VTG induction was confirmed by significant plasma VTG production following the exposure of FD-fed ovariectomized goldfish to a nominal estradiol-17β concentration of 100 μg/l for 31 days. Our data suggest that induction of plasma VTG using ovariectomized goldfish is a good tool for evaluating the estrogenic effects of endocrine-disrupting chemicals.

Combined biological and chemical assessment of estrogenic activities in wastewater treatment plant effluents.

Five wastewater treatment plant effluents were analyzed for known endocrine disrupters and estrogenicity. Estrogenicity was determined by using the yeast estrogen screen (YES) and by measuring the blood plasma vitellogenin (VTG) concentrations in exposed male rainbow trout (Oncorhynchus mykiss). While all wastewater treatment plant effluents contained measurable concentrations of estrogens and gave a positive response with the YES, only at two sites did the male fish have significantly increased VTG blood plasma concentrations after the exposure, compared to pre-exposure concentrations. Estrone (E1) concentrations ranged up to 51 ng L(-1), estradiol (E2) up to 6 ng L(-1), and ethinylestradiol (EE2) up to 2 ng L(-1) in the 90 samples analyzed. Alkylphenols, alkylphenolmonoethoxylates and alkylphenoldiethoxylates, even though found at microg L(-1) concentrations in effluents from wastewater treatment plants with a significant industrial content, did not contribute much to the overall estrogenicity of the samples taken due to their low relative potency. Expected estrogenicities were calculated from the chemical data for each sample by using the principle of concentration additivity and relative potencies of the various chemicals as determined with the yeast estrogen screen. Measured and calculated estradiol equivalents gave the same order of magnitude and correlated rather well (R(2)=0.6).

Reproductive and biochemical biomarkers in largemouth bass sampled downstream of a pulp and paper mill in Florida

The objective of this study was to evaluate the effects of bleached/unbleached kraft mill effluents (B/UKME) on the reproductive parameters of free-ranging Florida largemouth bass ( Micropterus salmoides floridanus). The reproductive parameters measured included gonadosomatic index (GSI), histological evaluation of gonads, and plasma concentrations of vitellogenin (VTG), 17 β-estradiol, and 11-ketotestosterone (11-KT). Hepatic ethoxyresorufin- O-deethylase (EROD) activity was measured as a marker of exposure to cytochrome P450-inducing agents in these effluents. Endpoints were compared among adult bass sampled from tributary and mainstream effluent-contaminated and reference sites. Females sampled from the site closest to the mill outfall had a significant five-fold increase in EROD activity compared to bass sampled from reference streams. Although sex hormones were significantly reduced in bass from exposed sites, there were no differences in VTG and GSI across sites. The absence of organism-level responses was probably not related to a lack of sensitivity, as previous studies in our laboratory have shown that bass exposed to these effluents exhibit changes in GSI and in other measures associated with reproductive success. In females, inverse relationships were observed between VTG and GSI and EROD activity. These relationship, however, were not consistent within all of the sites studied. Collectively, our findings indicate that hepatic EROD induction is an effective marker of B/UKME exposure in largemouth bass and that it might be associated with antiestrogenic effects in this species.

Plasma sex steroid and vitellogenin profiles during gonad development in wild Mediterranean amberjack ( Seriola dumerilii )

Mediterranean amberjacks, Seriola dumerilii Risso, were caught off the Pelagie Islands, in the south Mediterranean Sea, between May 1997 and June 1999. Fish blood was sampled, and gonads were collected at 10-day intervals throughout the spawning period and at monthly intervals during the resting period. Concentrations of plasma estradiol-17s (E2), testosterone (T), 17,20s-dihydroxy-4-pregnen-3-one (DHP) and vitellogenin (Vtg) in females; and plasma T and 11-ketoT (11-KT) in males were correlated with changes in gonadal development. The first females that had already ovulated (F5) were found in late May. Most mature females (F4) were caught in June. Post-spawned females (F6) were found from late July until September. Estradiol-17s was at baseline levels (<0.02 ng ml−1) during autumn/winter and rapidly peaked (6.29±0.68 ng ml−1) from May to early July. Plasma T levels showed a similar profile and were positively correlated to E2 (r 2=0.668) during the spawning period. Continuously elevated levels of E2 and T were observed during the spawning season in vitellogenic females (stages F3, F4 and F5). Resting females were found during the autumn/winter months. Vitellogenin levels increased during May and peaked in June, reverting to undetectable levels in August, in parallel with sex steroid changes. Plasma DHP levels peaked in June (283.45±97.3 pg ml−1), falling to basal values (<5 pg ml−1) in August. DHP levels were higher in mature females (F4) than in maturing (F3) and in partially ovulated (F5) females. DHP values increased during germinal vesicle migration, peaked during germinal vesicle breakdown and decreased again during complete oocyte hydration. In males, changes in T and 11-KT plasma levels were related to testis development. The highest levels of T (5.76±2.64 ng ml−1) were measured during spermatogenesis and highest 11-KT (5.28±3.6 ng ml−1) in males with milt, from May to June. This study provides information, for the first time, on the relation between plasma sex steroid profiles and gonad development in wild Mediterranean amberjack, a useful benchmark for broodstock monitoring under controlled conditions.

Route of exposure affects the oestrogenic response of fish to 4- tert-nonylphenol

When toxicants cause effects to aquatic organisms, it is often unclear by what route, or routes, the toxicant entered the affected organism. The toxicity of a compound depends on its degree of uptake, distribution and metabolism, as well as its molecular interactions at the site of action. It was hypothesised, that a hydrophobic chemical such as 4- tert-nonylphenol (4-NP), entering via the gills/skin, may be more oestrogenic than one entering through the diet, because in the latter case it will undergo metabolism in the small intestine and liver before entering the bloodstream. In this way, metabolism may reduce or eliminate the oestrogenic potential of 4-NP before it reaches target organs such as the gonads or liver. To compare the potency of 4- tert-nonylphenol when administered via different routes, male fathead minnows ( Pimephales promelas) were exposed to 4-NP either through waterborne exposure (experiment 1), or via the diet (experiment 2). Fish were exposed to 4-NP for 2 weeks either via the water at one of three nominal concentrations: 1, 10 or 50 μg/l (experiment 1) or 100, 500 or 1000 μg/day via the diet (experiment 2). Liver and blood samples were taken for vitellogenin mRNA and plasma vitellogenin quantification, respectively. Exposure of male fathead minnows to 50 μg/l of 4-NP in the water (experiment 1) and 500 and 1000 μg/day of 4-NP via the diet (experiment 2) induced vitellogenin mRNA. A similar pattern occurred for plasma vitellogenin induction, however, there was also a significant increase in plasma vitellogenin concentration in the fish exposed via the water to 10 μg/l of 4-NP. Using data from pharmacokinetics studies, an estimate for the total amount of 4-NP that entered the fish during each exposure was compared with the concentrations of plasma vitellogenin in each group of fish. The result showed a 10-fold greater sensitivity for 4-NP in fish exposed via the water compared with exposure via the oral route. Results obtained from this study indicate that a chemical such as 4-NP has a higher oestrogenic potential when it enters the bloodstream via the gills/skin of a fish compared with exposure through the diet.

Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent

It is now well established that there is a diverse array of chemicals discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wildlife groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological importance of endocrine disruption in wildlife. In this study, the effects of exposure to individual estrogenic chemicals (17α-ethinylestradiol; EE 2, bisphenol-A, and 4- tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE 2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout ( Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE 2 induced similar reproductive responses in the snails as in the fathead minnow ( Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 weeks exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. antipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates.

Estrogenic effects in flounder Platichthys flesus orally exposed to 4- tert-octylphenol

The estrogenic effect of dietary 4- tert-octylphenol in the flounder Platichthys flesus was investigated in the laboratory. In the first experiment male flounders were fed doses of 10, 50 and 100 mg 4- tert-octylphenol kg −1 BW or 0.05 mg 17β-estradiol kg −1 BW every second day for 10 days. Increases in the plasma content of vitellogenin were observed on day 6 by the first sampling in all treated groups. An effect of octylphenol on the size of the liver and testis was only seen in one of the exposed group (50 mg 4- tert-octylphenol kg −1 BW). An increase in the concentration of 4- tert-octylphenol was found in both liver and muscle tissues of all 4- tert-octylphenol treated fish. The concentration of 4- tert-octylphenol (ng g −1 WW) in liver tissue was four to five times greater than the concentration in muscle tissue. Only about 8% of the 4- tert-octylphenol administered to the group receiving 50 mg 4- tert-octylphenol kg −1 BW was retained in the liver and the muscles at the end of the experiment. In the second experiment male flounders were exposed to a low dose range of 4- tert-octylphenol (1 to 25 mg 4- tert-octylphenol kg −1 BW) in order to establish the dose-response relationship of 4- tert-octylphenol when applied by feeding to flounder. Vitellogenin induction (LOED) occurred at 10 and 5 mg 4- tert-octylphenol kg −1 BW after, respectively, 6 and 11 days of exposure. Based on a logistic regression analysis the effect dose (ED 50) could be determined as 8.2 and 5.6 mg 4- tert-octylphenol kg −1 BW on day 6 and 11, respectively. 4- tert-octylphenol accumulated in liver, muscle and testis tissue, and a significant accumulation in liver was found at 2.5 mg 4- tert-octylphenol kg −1 BW and in muscle and testis at 5 mg 4- tert-octylphenol kg −1 BW. Finally, a positive correlation between the plasma vitellogenin concentration and the liver tissue concentration of 4- tert-octylphenol was established as well as a positive correlation between the content of 4- tert-octylphenol in liver and muscle/testis tissue.