Development and validation of an analytical method for detection of estrogens in water.

An analytical procedure enabling routine analysis of four environmental estrogens at concentrations below 1 ng L(-1) in estuarine water samples has been developed and validated. The method includes extraction of water samples using solid-phase extraction discs and detection by gas chromatography (GC) with tandem mass spectrometry (MS-MS) in electron-impact (EI) mode. The targeted estrogens included 17alpha- and 17beta-estradiol (aE2, bE2), estrone (E1), and 17alpha-ethinylestradiol (EE2), all known environmental endocrine disruptors. Method performance characteristics, for example trueness, recovery, calibration, precision, accuracy, limit of quantification (LOQ), and the stability of the compounds are presented for each of the selected estrogens. Application of the procedure to water samples from the Scheldt estuary (Belgium - The Netherlands), a polluted estuary with reported incidences of environmental endocrine disruption, revealed that E1 was detected most frequently at concentrations up to 7 ng L(-1). aE2 was detected once only and concentrations of bE2 and EE2 were below the LOQ.

Multi-residue liquid chromatography/tandem mass spectrometry method for the detection of non-steroidal anti-inflammatory drugs in bovine muscle: optimisation of ion trap parameters.

A multi-residue liquid chromatography/tandem mass spectrometry method (LC/MS2) was developed for the detection of the non-steroidal anti-inflammatory drugs acetylsalicylic acid (via the marker residue salicylic acid), flunixin, phenylbutazone, tolfenamic acid, meloxicam and ketoprofen, in bovine muscle. After extraction of the bovine muscle with acetonitrile, the cleanup was performed using a Oasis HLB column. The evaporated eluate was reconstituted and analysed by LC/MS2. To obtain optimal detection of salicylic acid and phenylbutazone, the ion trap mass spectrometric parameters activation q and maximum ion injection time, respectively, were optimised. The activation q for salicylic acid was increased to obtain reliable detection of both salicylic acid and its product ion. The maximum ion injection time for the time segment containing phenylbutazone was decreased since there were not enough scans across the chromatographic peak of this compound. The multi-residue method was able to detect the different analytes below or at the maximum residue limit (MRL) or minimum required performance limit (MRPL) or, in the case of phenylbutazone and ketoprofen, at 100 and 20 microg kg(-1), respectively.

Testosterone and energy metabolism in the estuarine mysid Neomysis integer (Crustacea: Mysidacea) following exposure to endocrine disruptors.

A diverse set of reference compounds suspected of having an endocrine-disrupting mode of action (i.e., testosterone, flutamide, ethinylestradiol, precocene, nonylphenol, fenoxycarb, and methoprene) were tested for acute toxicity to the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Neomysis integer was very sensitive to all tested compounds, with 96-h median lethal concentrations in a narrow range between 0.32 and 1.95 mg/L. The pesticides methoprene and fenoxycarb, both synthetic insect juvenile hormone analogs, were most toxic to N. integer. In addition, the short-term sublethal effects of methoprene and nonylphenol (an estrogen agonist) on the energy and steroid metabolism of N. integer were evaluated. Both compounds significantly affected energy and testosterone metabolism of N. integer at concentrations below acute toxicity levels. Energy consumption in methoprene- and nonylphenol-exposed mysids was significantly induced at 100 microg/L, resulting in a lower cellular energy allocation in these animals. Testosterone phase I metabolism was affected at 10 microg/L, whereas glycosylation was the most important phase II pathway affected in mysids exposed to 100 microg/L of both compounds. Methoprene exposure resulted in a concentration-dependent increase in the metabolic androgenization ratio. Mysids exposed to nonylphenol at 10 microg/L had a significantly higher metabolic androgenization ratio. The present study indicates that energy and testosterone metabolism of mysids, as endpoints, are able to detect endocrine-disruptive activity of chemicals after short-term exposure to environmentally realistic levels of endocrine disruptors.

Testosterone metabolism in the estuarine mysid Neomysis integer (Crustacea; Mysidacea) following tributyltin exposure.

Current evidence suggests that the biocide tributyltin (TBT) causes the development of imposex, a state of pseudohermaphrodism in which females exhibit functional secondary male characteristics, by altering the biotransformation or elimination of testosterone. Imposex in gastropods following TBT exposure is the most complete example of the effects of an endocrine disrupter on marine invertebrates. Previous studies have demonstrated that the estuarine mysid Neomysis integer converts testosterone into multiple polar and nonpolar metabolites resulting from both phase I and phase II biotransformations. In this study, the effects of TBT chloride (TBTCl) on the phase I and II testosterone metabolism of N. integer were evaluated. The TBTCl was highly toxic to N. integer (96-h median lethal concentration [LC50] of 164 ng/L). To assess the effects on testosterone metabolism, mysids were exposed for 96 h to different concentrations of TBTCl (control, 10, 100, and 1,000 ng/L), and testosterone elimination as polar hydroxylated, nonpolar oxido-reduced, and glucose- and sulfate-conjugated metabolites was examined. The TBTCl differentially affected testosterone metabolism. The effect of TBTCl on phase I metabolism was unclear and has been shown to vary among species, likely depending on the inducibility or presence of certain P450 isozyme families. Reductase activity and metabolic androgenization were induced in the 10-ng/L treatment, whereas higher concentrations resulted in a reduction of sulfate conjugation. The exact mechanisms underlying TBT-induced imposex and alterations in the steroid metabolism need to be further elucidated.

Simultaneous determination of different antibiotic residues in bovine and in porcine kidneys by solid-phase fluorescence immunoassay.

Parallux, a solid-phase fluorescence immunoassay (SPFIA) developed for antibiotic residue detection in milk, was used for analysis of bovine and porcine kidney tissue. Four tetracyclines, 2 broad-spectrum cephalosporins, 3 beta-lactam antibiotics, and cephapirin were detected in one run after minimal sample preparation. This commercially available test system is designed as cartridges, each with a combination of 1-4 tests. One cartridge can be used to detect 4 analytes in the same sample, or 1 or 2 analytes in different samples. The cartridge with the combination tetracyclines-ceftiofur-penicillin-cephapirin was selected because tetracyclines, beta-lactam antibiotics as well as cephalosporins, are registered for oral or parenteral use in bovines and pigs in Europe. The test is qualitative and is recommended only for screening. Tetracycline, oxytetracycline, chlortetracycline, and doxycycline were easily detected at 300 ppb with the tetracyclines channel; ceftiofur at 1000 ppb and cefquinome at 200 ppb with the ceftiofur channel; penicillin G, ampicillin, and amoxicillin at 50 ppb with the penicillin channel; and cephapirin at 100 ppb with the cephapirin channel. These levels are equal to or lower than the corresponding maximal residue limits in kidney tissue. Cephalexin was not detected. The SPFIA test can be used as an alternative to classical inhibition tests and for post-screening inhibitor- positive kidneys, because it detects 3 specific groups of antibiotics, which enables selection of specific confirmatory methods for identification and quantification.

Analysis on residues of estrogens, gestagens and androgens in kidney fat and meat with gas chromatography-tandem mass spectrometry.

The use of estrogens, gestagens and androgens (EGAs) in animal fattening is prohibited in the European Community. Based on the general detection capabilities of Belgian laboratories, National Minimum Required Performance Limits (National MRPLs) for a number of EGAs have been imposed by the inspection services. Selective hyphenated techniques, e.g. GC-MS and GC-MS2, with high detection capability are needed. Beta-trenbolone, which is meant to be a "problem" molecule for GC-MS, can be detected at the 2 microg/kg level using GC-MS2. Based on the National MRPLs in different matrices, our laboratory has divided the EGAs into a class system. In this set-up, analysis of EGAs in kidney fat and meat is discussed.

Testosterone metabolism in the estuarine mysid neomysis integer (Crustacea; Mysidacea): identification of testosterone metabolites and endogenous vertebrate-type steroids.

Testosterone metabolism by Neomysis integer (Crustacea; Mysidacea) was assessed to obtain initial data on its metabolic capacity. N. integer were exposed to both testosterone and [(14)C]testosterone. Identification of testosterone metabolites and endogenous steroids was performed using thin-layer chromatography and liquid chromatography with multiple mass spectrometry. Endogenous production of testosterone in mysids was detected for the first time. N. integer were exposed to testosterone and metabolized administered testosterone extensively. At least 11 polar testosterone metabolites (R(f,metabolite) < R(f,testosterone)), androstenedione, dihydrotestosterone, and testosterone were produced in vivo by N. integer. A sex-specific testosterone metabolism was also observed, although this observation requires further confirmation. The anabolic steroid beta-boldenone was also identified for the first time in invertebrates. The metabolic pathway leading to the formation of beta-boldenone remains unknown, since the steroidal precursor androstadienedione could not be detected. These results reveal interesting similarities in enzyme systems in invertebrate and vertebrate species. Alterations in steroid hormone metabolism may be used as a new biomarker for the effects of endocrine disruptors in invertebrates.