Combining passive samplers and biomonitors to evaluate endocrine disrupting compounds in a wastewater treatment plant by LC/MS/MS and bioassay analyses.

Two types of integrative sampling approaches (passive samplers and biomonitors) were tested for their sampling characteristics of selected endocrine disrupting compounds (EDCs). Chemical analyses (LC/MS/MS) were used to determine the amounts of five EDCs (nonylphenol, bisphenol A, estrone, 17beta-estradiol and 17alpha-ethinylestradiol) in polar organic chemical integrative samplers (POCIS) and freshwater mussels (Unio pictorum); both had been deployed in the influent and effluent of a municipal wastewater treatment plant (WWTP) in Genoa, Italy. Estrogenicity of the POCIS samples was assessed using the yeast estrogen screen (YES). Estradiol equivalent values derived from the bioassay showed a positive correlation with estradiol equivalents calculated from chemical analyses data. As expected, the amount of estrogens and EEQ values in the effluent were lower than those in the influent. Passive sampling proved to be the preferred method for assessing the presence of these compounds since employing mussels had several disadvantages both in sampling efficiency and sample analyses.

Plasma steroids in mature common dentex (Dentex dentex) stimulated with a gonadotropin-releasing hormone agonist.

The aim of this study was to identify the major C21 steroids produced in vivo during artificially induced final oocyte maturation and spawning in female common dentex (Dentex dentex). During the spawning season, mature females were treated with a gonadotropin-releasing hormone agonist (GnRHa)-loaded delivery system, with or without pimozide (given as a single dose at the beginning of the experiment). Blood samples were collected at various intervals during the experiment and were assayed for GnRHa, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P). A higher percentage of ovulated females was observed in GnRHa-implanted fish, which produced over 10 times more eggs than controls. Relative fecundity was highest in the GnRHa + pimozide group and lowest in controls. The viability of naturally released eggs was low (2 to 15%) in all groups. Plasma concentrations of 17,20beta-P in GnRHa-implanted fish did not increase, but those in control fish decreased, such that there was a significant difference between control and treated fish between 2 and 10 days after treatment. In another experiment, ovulating common dentex were injected intramuscularly with a single dose of 50 microg kg(-1) of GnRHa in saline and were sampled for blood at 0, 3, 6, 12, and 24 h postinjection. A single water sample was taken from the tanks at 9 h postinjection, the tanks having been emptied and refilled at 6 h. Measurements were made of plasma and water concentrations of free and conjugated 17,20beta-P, 17,20beta,21-P, 17beta-oestradiol (E2), and GnRHa (plasma only). The GnRHa injection increased plasma levels of all steroids, with free 17,20beta-P reaching maximal levels within 3 h. GnRHa treatment also increased the amounts of free and conjugated steroids released into the water between 6 and 9 h.

Gonadotrophin-releasing hormone agonist stimulates milt fluidity and plasma concentrations of 17,20beta-dihydroxylated and 5beta-reduced, 3alpha-hydroxylated C21 steroids in male plaice (Pleuronectes platessa).

Spermiating male plaice were caught in the North Sea and acclimatised to laboratory conditions. In two experiments, males were injected intramuscularly with either microspheres or pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Blood was sampled at 2- to 5-day intervals. Individual blood plasma specimens were assayed for testosterone, 5beta-reduced, 3alpha-hydroxy ("5beta,3alpha") steroids and sulphated 17, 20beta-dihydroxy ("17,20beta") steroids. Pooled plasma samples were also assayed for free and sulphated 17, 20beta-dihydroxy-4-pregnen-3-one, free 11-ketotestosterone, and glucuronidated testosterone and 11-ketotestosterone. Plasma concentrations of all steroids were significantly elevated by GnRHa from 2 to 5 days onwards following treatment. The most marked changes occurred in the concentrations of the sulphated 17,20beta steroids, which comprised approximately equal amounts of 5beta-pregnane-3alpha,17,20beta-triol 20-sulphate (3alpha,17, 20beta-P-5beta-S) and 5beta-pregnane-3beta,17,20beta-triol 20-sulphate, rising from ca. 1 to 30-80 ng/ml in the first and from ca. 8 to 80 ng/ml in the second experiment. Concentrations of 5beta, 3alpha steroids matched those of 17,20beta steroids in one experiment. However, in the other experiment, the two RIAs yielded highly disparate results in about 50% of the fish (including males in the control group). The plasma of these fish contained excessive amounts of 5beta,3alpha-immunoreactive material between 10 and 25 days. This material was identified as 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one 21-sulphate (a metabolite of 11-deoxycortisol). All previous studies have indicated that when plasma concentrations of this steroid are high, so are those of 3alpha,17,20beta-P-5beta-S. This is the first indication that these steroids are regulated independently. In a third experiment, milt fluidity and production were assessed at 10, 15, and 25 days following GnRHa implantation. Milt volume and fluidity were significantly enhanced by the GnRHa treatment.

Changes in plasma gonadotropin II and sex steroid hormones, and sperm production of striped bass after treatment with controlled-release gonadotropin-releasing hormone agonist-delivery systems.

Spermiating striped bass, a perciform fish, were treated with two controlled-release gonadotropin-releasing hormone agonist (GnRHa)-delivery systems, and the resulting changes in plasma gonadotropin II (GTH II), testosterone (T), 11-ketotestosterone (11-KT), 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), and 17,20beta,21-trihydroxy-4-pregnen-3-one (17,20beta,21-P) were correlated with changes in milt production and sperm density. GnRHa-delivery systems induced a sustained elevation of plasma GnRHa and GTH II for 14 days. Plasma T levels were unchanged after GnRHa treatment, while 11-KT levels increased significantly. Plasma 17,20beta-P also increased after GnRHa treatment and remained elevated compared to levels in controls, while plasma 17,20beta,21-P levels were unaffected. Both GnRHa-delivery systems induced many-fold increases in total expressible milt, lasting throughout the 14 days of the study. Sperm density decreased 2 days after GnRHa treatment, with a subsequent increase by Day 7. This study demonstrates that GnRHa-delivery systems induce a sustained elevation of plasma GTH II levels in striped bass, resulting in a long-term enhancement of milt production. The endocrine changes observed suggest that 11-KT and 17,20beta-P, but not 17,20beta,21 -P, are mediating the effects of GTH II on spermiation.

Use of a radioimmunoassay which detects C21 steroids with a 5beta-reduced, 3alpha-hydroxylated configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

Two radioimmunoassays (RIAs) have been developed which detect C21 (pregnane) steroids with a 5beta-reduced, 3alpha-hydroxyl (5beta, 3alpha) configuration. One RIA only detects 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one and 3alpha, 17-dihydroxy-5beta-pregnan-20-one, whilst the other detects a range of 5beta,3alpha steroids, including 5beta-pregnane-3alpha,17,20 beta-triol, a major metabolite of 17, 20beta-dihydroxy-4-pregnen-3-one, the putative oocyte maturation-inducing steroid in plaice Pleuronectes platessa. The RIAs, in conjunction with reverse-phase high-performance liquid chromatography (HPLC), have identified and quantified the steroids in plasma and urine of reproductively mature females. Total levels of 5beta,3alpha metabolites which can be extracted with diethyl ether (i.e., free steroids) are relatively low (<10 ng/ml). However, total levels of 5beta,3alpha metabolites released by solvolysis (i.e. , sulphated steroids) are very high (up to 1000 ng/ml in plasma and 20 microg/ml in urine). On HPLC, these metabolites have been identified (in order of their abundance in plasma) as: 3alpha,17, 21-trihydroxy-5beta-pregnan-20-one, 5beta-pregnane-3alpha,17, 20beta-triol, 5beta-pregnane-3alpha,17,20alpha-triol, 3alpha,11beta, 17,21-tetrahydroxy-5beta-pregnane-20-one, and 3alpha, 17-dihydroxy-5beta-pregnan-20-one. Levels of the first three steroids are significantly elevated in female plaice undergoing natural or gonadotrophin-induced final oocyte maturation.

Use of a radioimmunoassay which detects C21 steroids with a 17, 20beta-dihydroxyl configuration to identify and measure steroids involved in final oocyte maturation in female plaice (Pleuronectes platessa).

A radioimmunoassay (RIA) has been developed to detect a range of C21 (pregnane) steroids with a 17,20beta-dihydroxyl (17,20beta) configuration. In conjunction with reverse-phase high-performance liquid chromatography (HPLC), it identifies and quantifies the metabolites of 17,20beta-dihydroxy-4-pregnen-3-one, the putative "maturation-inducing steroid" in female plaice Pleuronectes platessa. Total levels of 17,20beta metabolites which can be extracted from plasma or urine with diethyl ether (i.e., free steroids) are very low (<3 ng/ml). However, total levels of 17,20beta metabolites which can be released by solvolysis (i.e., sulphated steroids) are very high (up to 1 microg/ml in plasma and 10 microg/ml in urine). On HPLC, these sulphated metabolites have been identified (in order of abundance in plasma) as: 5beta-pregnane-3alpha,17,20beta-triol, 5beta-pregnane-3beta,17,20beta-triol, 17, 20beta-dihydroxy-4-pregnen-3-one, and 17, 20beta-dihydroxy-5beta-pregnan-3-one. These steroids are absent from plasmas of fish which have not yet begun final oocyte maturation. The results support the hypothesis that 17, 20beta-dihydroxy-4-pregnen-3-one is the maturation-inducing steroid in plaice but that it is rapidly metabolised to render it inactive. The results also show that the '17,20beta'-RIA, in combination with an overnight acid solvolysis procedure, is a useful procedure for monitoring the effects of exogenous factors (such as gonadotrophin injections) on final oocyte maturation in female plaice.

Excretion of free and conjugated steroids in rainbow trout (Oncorhynchus mykiss): evidence for branchial excretion of the maturation-inducing steroid, 17,20 beta-dihydroxy-4-pregnen-3-one.

The goal of this study was to identify excretory routes of three main steroids produced by sexually mature male and female rainbow trout: 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P), sulfated 17,20 beta-P (17,20 beta-P-S), and testosterone glucuronide (TG). Spermiating males or maturing trout were cannulated via the dorsal aorta and urinary bladder and injected with tritiated steroids. Blood, water, and urine were sampled over the next 12 hr when the fish were killed and bile was collected. The identities of the excreted products were determined by anion-exchange chromatography, reverse-phase high-performance liquid chromatography, enzyme hydrolysis, acid solvolysis, microchemical modification, and thin-layer chromatography. Following the injection of tritiated 17,20 beta-P, 25% of the radioactivity rapidly appeared unmodified in the water; 15% appeared slowly in the urine, mainly as 17,20 beta-P-S; and 40% was recovered in the bile, mainly as 17,20 beta-P-glucuronide. 17,20 beta-P was shown to be released into the water via the gills. Over the 12-hr sampling period, 20% of the 17,20 beta-P released into the water was taken up again by the fish (also branchially). A mathematical analysis showed that 40% of the 17,20 beta-P would have been released into the water in the absence of uptake. Following the injection of tritiated 17,20 beta-P-S, 63% appeared very rapidly, in an unmodified form, in the urine, and 15% was recovered in the bile. Following the injection of tritiated TG, 9% appeared slowly, mainly untransformed, in the urine, and 59% was recovered in the bile. These results show that the three types of steroids are released into the water by three different routes: free steroids, gills; sulfated steroids, urine; and glucuronidated steroids, bile.