Estrogenic activity and contributing compounds in stagnant water bodies with massive occurrence of phytoplankton.

Stagnant water bodies have generally received little attention regarding the presence of endocrine disruptive compounds, although they can integrate diverse pollutants from multiple different sources. Many compounds of anthropogenic as well as natural origin can contribute to the overall estrogenicity of surface waters and some of them can exhibit adverse effects on aquatic biota even in very low concentrations. This study focused on freshwater ponds and reservoirs affected by water blooms and determined the estrogenic activity of water by in vitro bioassay as well as concentrations of several important groups of estrogenic compounds (estrogenic hormones, alkylphenols, and phytoestrogens) by LC-MS/MS analyses. Estrogenic hormones were found at concentrations up to 7.1 ng.L-1, similarly to flavonoids, whose concentrations did not exceed 12.5 ng.L-1. Among alkylphenols, only bisphenol A and 4-tert-octylphenol were detected in levels reaching 100 ng.L-1 at maximum. Estrogenic activity of water samples varied from below the quantification limit to 1.95 ng.L-1. There does not seem to be any general causal link of the massive phytoplankton occurrence with the estrogenicity of water or concentration of phytoestrogens, since they showed no direct relationship with the phytoplankton abundance or composition across sites. The contribution of the analysed compounds to the estrogenic activity was calculated in three scenarios. In minimum scenario, just the compounds above quantification limit (LOQ) were taken into account and for most samples, only minor part (<6%) of the biological activity could be explained. In the mean and maximum scenarios, we included also compounds below LOQ into the calculations at the level of LOQ/2 and LOQ, respectively. In these cases, a considerable part of the estrogenic activity could be attributed to the possible presence of steroid estrogens below LOQ. However, for the samples with estrogenic activity greater than 1 ng.L-1, more than 50% of the estrogenic activity remained unexplained even in the maximum scenario. Probably other compounds or possible interactions between individual substances cause the estrogenic activity in these types of water bodies and in this case, the results of LC-MS/MS analyses cannot sufficiently predict the biological effects. A complex approach including bioassays is needed when assessing the estrogenicity of these types of surface waters.

Retinoid-like compounds produced by phytoplankton affect embryonic development of Xenopus laevis.

Teratogenic effects, which were remarkably similar to those induced by retinoic acids, have been seen in wild frogs indicating possible source of retinoids in the environment. Recent studies indicate that some cyanobacterial species can contain teratogenic retinoic acids (RAs) and their analogues. Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis. The study investigated the effects of exudates (extracellular compounds) of two cyanobacteria species with retinoic-like activity and one algae species on embryonic development of amphibians. The retinoid-like activity determined by in vitro reporter gene assay reached 528ng retinoid equivalents (REQ)/L and 1000ng REQ/L in exudates of Cylindrospermopsis raciborskii and Microcystis aeruginosa, respectively, while algal exudates showed no detectable activity. Total mean of retinoid-like copounds into exudate was 35.6ng ATRA/mil.cells for M.aeruginosa and 6.71ng ATRA/mil.cells for C.raciborskii, respectively. Toxicity tests with amphibian embryos up to 96h of development were carried out according to the standard guide for the Frog Embryo Teratogenesis Assay Xenopus. Lowest observed effect concentrations (LOEC) of malformations (2.5-2.6µg/L REQ) were two times lower than LOEC for ATRA (5µg/L). The exudates of both cyanobacteria were indeed provoking diverse teratogenic effects (e.g. tail, gut and eyes deformation) and interference with growth in frogs embryos, while such effects were not observed for the algae. Xenopus embryos were also exposed to all-trans retinoic acid (ATRA) in concentration range (1-40µg/L) equivalent to the REQs detected in cyanobacterial exudates. ATRA (10µg/L) caused similar teratogenic phenotypes at corresponding REQs as cyanobacterial exudates. The study confirms the ability of some species of cyanobacteria to produce retinoids naturally and excrete them directly into the environment at concentrations which might have adverse influence on the development of amphibians.

[Sensitivity of GC-MS in the detection of benzodiazepines in the urine in the form of trimethylsilyl derivatives]. / Citlivý GC-MS prukaz benzodiazepinu v moci ve forme jejich trimethylsilylderivátu.

A sensitive evidence of trace concentrations of benzodiazepines and their metabolites in urine can be enabled after special sample preparation including enzymatic hydrolysis, special solid phase extraction, silylation and following analysis by gas chromatography with mass spectrometry in electron impact mode. After optimalization of the procedure the extraction recovery values in the range 50-85% are achieved. The scale of retention times with basic mass spectral data are presented for the spectrum of silylated benzodiazepines which can overlap some gaps in the standard toxicological literature up to now available.

[Detection of diacetylmorphine (heroin) use by thin-chromatography analysis of urine]. / Dukaz aplikace diacetylmorfinu (heroinu) rozborem moce metodou tenkovrstevné chromatografie.

The proof of diacetylmorphine (heroin) application is based on the identification of its specific metabolite 6-monoacetylmorphine simultaneously detected with the main metabolite morphine in human urine. Codeine is another morphine derivative appearing in cases of diacetylmorphine abuse; it can be considered a metabolite of 6-acetylcodeine, a typical impurity found in raw heroin. An analytical procedure for detection of the mentioned morphine basesin urine is presented, including a screening method. A careful extraction method is required for 6-monoacetylmorphine. To classify it into the code system of the screening, its properties are expressed by a threecomponent code. Subsequent identity confirmation of the mentioned bases by means of thin layer chromatography uses mobile phases, in which optimal separation effects are achieved, even in the presence of nicotine or caffeine and its metabolites theobromine and theophylline. Circumstances of 6-monoacetylmorphine discovery in urine are discussed.

High-performance liquid chromatographic confirmation of cocaine and benzoylecgonine in biological samples using photodiode-array detection after toxicological screening.

In the later stages after intake, the important markers of cocaine abuse are its main metabolites in urine, benzoylecgonine and ecgonine methyl ester. The efficiency of the extraction of amphoteric benzoylecgonine together with cocaine from aqueous media by means of various solvents at various pH values and by means of a mixed solid phase was tested. The extraction of benzoylecgonine with diethyl ether is not efficient, whereas chloroform, dichloromethane or mixed solid-phase extraction give satisfactory results. The analytical strategy for the general chromatographic screening and identification of unknown drugs in biological samples based on diethyl ether extraction was modified to permit the sensitive detection of cocaine abuse also on the basis of benzoylecgonine. A complementary high-performance liquid chromatographic method with photodiode-array detection after solid-phase extraction was introduced for specific confirmation and determination of cocaine and benzoylecgonine.

Systematic analytic procedure for identification of unknown medicaments in biological material.

The presented method has been used in toxicological practice for identification of unknown medicaments in biological material longer than fifteen years with good results. This method described as TLC/CR-screening is a combination of thin layer chromatography (TLC) with a system of colour reactions (CR-screening), the principle being evaluation of characteristic features (qualities) of each detected unknown substance (parent medicament form or its metabolite) from three points of view: the type of ionization during the extraction, the mobility in basic TLC-system in comparison with five reference standards, the reactivity with system reagents for detection. The properties of each detected substance expressed by adequate symbols are put together into three component codes, under which corresponding standards of medicaments and/or their metabolites can be found in the library of code system.