High enrichment factors in chemical analysis of progestins and in bioassays: insights beyond trace levels.

Concerns are growing about adverse effects of progestins on biota, even at ultra-trace concentrations. The enrichment factor (EF) from extraction of analytes in environmental samples that is needed for sample pre-concentration can affect not only performance of the analytical method but also the matrix effect. Therefore, the present study aimed to assess the influence of high sample EF on performance of the high-performance liquid chromatography with atmospheric pressure chemical ionization and photoionization coupled with high-resolution mass spectrometry (HPLC-APCI/APPI-HRMS) method for analysis of progestins in waste water treatment plant (WWTP) effluents and surface waters and analysis of (anti-)progestogenic activities measured by (anti-)PR-CALUX bioassays. The results showed that HPLC-APCI/APPI-HRMS coupled with solid-phase extraction and a high EF (33,333 Lwater/Lextract) enabled the detection of more compounds compared to samples with lower sample EF (10,000 Lwater/Lextract). The matrix effect did not increase proportionally compared to lower EFs (10,000 and 16,666 Lwater/Lextract), and lower limits of quantification were achieved in WWTP effluents and surface waters. The results of bioassays have shown that relative EF of 25 Lwater/Lbioassay appears high enough to detect progestogenic activity in treated waste water. Our study is one of the first to provide insights into sample pre-concentration in analysis of progestins and progestogenicity in aquatic environments.

Identification of hot spots and co-occurrence patterns of activities on thyroid hormone receptor and transthyretin binding in passive samplers from Czech surface waters.

One of the less studied in vitro biological activities in the aquatic environment are thyroid hormone receptor beta (TRß)-mediated agonistic and antagonistic activities and transthyretin (TTR) binding activity. They were measured mostly using active sampling methods, but rarely found. It is unclear if these activities co-occur, and the drivers of the (anti-)TRß activity are mostly unknown. Therefore, the main aim of the study was to determine (anti-)TRß activities as well as transthyretin (TTR) binding activity in passive samplers from Czech surface waters in combination with the search for the effect drivers based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis by applying suspect screening. Passive samplers (polar organic chemical integrative samplers, POCIS) were deployed at twenty-one sites (all ends of watersheds and other important sites in Elbe River) in the Czech rivers. The (anti-)TRß and TTR binding activity were measured using (anti-)TRß-CALUX and TTR-TRß-CALUX bioassays. Anti-TRß activity was found at eight sites, and TTR binding activity co-occurred there at six of these sites. The co-occurrence of TRß-mediated antagonistic activity and TTR binding indicate that they may have common effect drivers. No sample exhibited TRß agonistic activity. The extract from the site Bílina River, the most burdened with anti-TRß activity, was further successfully fractionated, and this activity was revealed in the fraction, where mid-polar compounds prevailed. However, the suspect LC-HRMS analysis did not reveal the chemical effect drivers. Our results showed that anti-TRß activity can be found in surface waters by employing passive sampling and frequently co-occurs with TTR binding activity. Overall, the fractionation procedure and non-target data acquisition used in this study can serve as a basis for searching the effect drivers in future research.

Organic UV filters: Occurrence, risks and (anti-)progestogenic activities in samples from the Czech aquatic environment and their bioaccumulation in fish.

The occurrence, environmental risks and contribution of organic UV filters to detected (anti-)progestogenic activities were examined in samples of wastewater treatment plant influents and effluents, various surface waters and fish from the Czech Republic. Of the 20 targeted UV filters, 15 were detected in the WWTP influent samples, 11 in the effluents, and 13 in the surface water samples. Benzophenone-3, benzophenone-4, and phenyl benzimidazole sulfonic acid (PBSA) were found in all water samples. Octocrylene, UV-327 and 4-methylbenzylidene camphor exceeded the risk quotient of 1 at some sites. In the anti-progestogenic CALUX assay, 10 out of the 20 targeted UV filters were active. Anti-progestogenic activities reaching up to 7.7 ng/L, 3.8 ng/L, and 4.5 ng/L mifepristone equivalents were detected in influents, effluents, and surface waters, respectively. UV filters were responsible for up to 37 % of anti-progestogenic activities in influents. Anti-progestogenic activities were also measured in fish tissues from the control pond and Podrouzek (pond with the highest number of detected UV filters) and ranged from 2.2 to 9.5 and 1.9 to 8.6 ng/g dw mifepristone equivalents, respectively. However, only benzophenone was found in fish, but it does not display anti-progestogenic activity and thus could not explain the observed activities.

Effects of mifepristone, a model compound with anti-progestogenic activity, on the development of African clawed frog (Xenopus laevis).

The objective of this study was to assess the effects of a model substance with anti-progestogenic activity on development of African clawed frog (Xenopus laevis) from tadpole to juvenile stage. Mifepristone, a synthetic progesterone receptor-blocking steroid hormone used in medicine as an abortifacient, was chosen as a model compound with anti-progestogenic activity. In the experiment, African clawed frog tadpoles were exposed to mifepristone at three concentrations (2, 21, and 215 ng L-1). A control group was exposed to dimethyl sulfoxide (DMSO; 0.001 %). The experiment started when tadpoles reached stages 47-48 according to Nieuwkoop and Faber (NF; 1994) and continued until stage NF 66, when metamorphosis was complete. Exposure to mifepristone had no significant effect on the rate of tadpole development, occurrence of morphological anomalies, weight, body length, or sex ratio. Mortality was within an acceptable range of 0-3.6 % throughout the test and did not differ among the groups. Histopathological examination of the gonads and thyroid gland revealed no significant changes. Therefore, we can conclude that mifepristone had no negative effect on development of the African clawed frog up to juvenile stage. Nevertheless, at the highest tested mifepristone concentration (215 ng L-1), gene expression analysis revealed up-regulation of mRNA expression of nuclear progesterone receptor (npr), membrane progesterone receptor (mpr), estrogen receptor beta (esrß), and luteinizing hormone (lh) in the brain-pituitary complex of exposed frogs at stage NF 66. Higher mRNA expression of npr was also found in frogs exposed to 22 ng L-1 mifepristone compared to the solvent control. These findings confirmed the anti-progestogenic activity of mifepristone in frogs because the up-regulation of progesterone receptors occurs if progesterone availability in the body is reduced. All the observed changes in combination may have negative consequences for reproduction and reproductive behavior later in life.

Clinical and Laboratory Parameters of Carp Edema Virus Disease: A Case Report.

In the present study, we describe a natural outbreak of carp edema virus disease (CEVD) in koi carp, concentrating on clinical manifestation, gross and microscopic pathology, immunological parameters, viral diagnostics, and phylogenetic analysis. Examination of white blood cell parameters showed increased monocyte and decreased lymphocyte counts in CEV-affected fish compared to healthy control fish. Regarding immune system functioning, the present work shows, for the first time, enhanced phagocytic activity in CEV-affected fish. Respiratory burst of phagocytes was strongly increased in diseased fish, the increase being attributed to an increased phagocyte count rather than enhancement of their metabolic activity. The present work also newly shows histopathological changes in the pancreatic tissue of diseased koi.

Environmental water extracts differentially activate zebrafish and human nuclear progesterone receptors.

Many reports on anti-progestogenic activities in aquatic environments have been published in the past decade. These are monitored mainly by in vitro reporter gene bioassays based upon the human progesterone receptor (PR). However, results obtained by some human in vitro bioassays may not be relevant for aquatic animals, especially fish. The present work aimed to detect fish (anti-)PR activity in waste- and receiving surface waters. In parallel, human (anti-)PR activity was analysed to determine if there was any connection between human and fish (anti-)PR activities. Finally, (anti-)PR activities were linked to the occurrence of progestins in water samples. Human PR agonistic activity was detected in all wastewater and most receiving surface water samples. Nevertheless, zebrafish PR (zfPR) agonistic activity was found in only two influent wastewater samples (max. 117 ng/L 17α,20ß-dihydroxy-4-pregnen-3-one [DHP] equivalents). Analysed synthetic progestins and progesterone accounted for 14 % to 161 % of detected human PR (hPR) agonistic activity in water samples. Progesterone also contributed significantly to zfPR agonistic activity (up to 10 %) in raw wastewater. The anti-hPR activity was detected also in most wastewater and some surface water samples, but synthetic progestins did not trigger anti-zfPR activity in excess of LOQ values. In addition, altrenogest, dienogest, and ulipristal acetate were tested for their potency to zfPR for the first time. The activity analyses of both pure substances and environmental samples showed that human and zebrafish progesterone receptors are differentially activated. Therefore, results based on human PR in vitro bioassays could not predict fish PR activities in the environment.

Effects of the synthetic progestin levonorgestrel on some aspects of thyroid physiology in common carp (Cyprinus carpio).

The objective of the present study was to assess the effects of levonorgestrel (LNG), a synthetic progestin, on early development and the thyroid system of carp using morphological, histological, immunohistochemical, and gene expression analysis. Fish were exposed to LNG at three levels (3, 31, and 310 ng L-1) from eggs to the onset of juvenile stage (47 days). LNG had no significant effect on early development in common carp or on the occurrence of morphological anomalies. No pathological alterations of the thyroid follicles were found. Immunohistochemical examination of the thyroid follicles using antibodies against thyroxin did not show any differences in fish exposed to 310 ng L-1 LNG compared to the controls. mRNA expression of iodothyronine deiodinases (dio1, 2, 3) was differentially affected by LNG treatment during carp development. Most importantly, dio3 was markedly downregulated in fish exposed to all three LNG levels compared to the controls at the conclusion of the experiment (47 days post-fertilization). A decrease in dio1 or dio3 or an increase in dio2 transcription observed at different time points of the study may be a sign of hypothyroidism. mRNA expression of genes npr, esr1, and esr2b in the body and npr and esr2b in the head of fish exposed to 310 ng L-1 LNG was significantly upregulated compared to the solvent control group at the end of the test. Together, these results show that levonorgestrel caused parallel changes in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-gonad axes.

Octinoxate as a potential thyroid hormone disruptor - A combination of in vivo and in vitro data.

Ultraviolet filters are commonly used in various cosmetic products. Due to their huge consumption ultraviolet filters become a part of the environment. Octinoxate is a commonly used ultraviolet filter that is widely detected in the aquatic environment. In our study, we investigated whether this ultraviolet filter is able to disrupt thyroid hormone regulation after six weeks of exposure in rainbow trout (Oncorhynchus mykiss). Thyroid hormones play crucial role in development and regulation of the organism and its disruption could cause the whole-body imbalance. Our study includes a compilation of in vivo and in vitro tests. The results of the in vivo experiment revealed a significant increase in thyroxine hormone in plasma for the highest tested dose of octinoxate (i.e. 395.6 µg/kg). We examined selected tissues (liver and cranial kidney) to determine the mRNA expression of genes involved in thyroid hormones regulation. The analysis confirmed downregulation of deiodinase 2 mRNA expression for the highest tested dose (i.e. 395.6 µg/kg) and downregulation of paired box 8 mRNA for medium (96 µg/kg) and the highest octinoxate dose (395.6 µg/kg.) only in cranial kidney. In vitro analysis indicated that octinoxate does not elicit (anti-)thyroid activity via thrß and does not behave as a transthyretin ligand. Based on our results, octinoxate has a potential to act as a thyroid hormone disruptor, but further research required to better understand the entire regulatory mechanism.

Immunohistochemical investigation of epithelial, mesenchymal, neuroectodermal, immune and endocrine markers in sterlet (Acipenser ruthenus), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio).

Immunohistochemistry (IHC) is a laboratory method widely used to characterize tissue and cell origin, both in human and veterinary medicine. In fish, however, little is known about staining characteristics of most tissue types, and especially for less studied chondrostean fish. The aim of this study was to examine the specificity of various immunohistochemical markers in tissues of chondrostean and teleostean fish and to validate diagnostic tests. Sterlet (Acipenser ruthenus L.), shortnose sturgeon (Acipenser brevirostrum) and common carp (Cyprinus carpio L.) were examined. Markers were chosen as representatives of epithelial (cytokeratin AE1/AE3), mesenchymal (vimentin), neuroectodermal (S-100 protein), lymphoid (leukocyte common antigen, LCA) and endocrine (thyroglobulin, thyroxin) tissues and organs. Applied antibodies were of monoclonal or polyclonal mammalian origin and primarily intended for human medicine research or diagnostic application. No species differences were obvious while examining sterlet, shortnose sturgeon and carp. Cytokeratin AE1/AE3, vimentin, S-100 protein and thyroxin were positive on targeted tissues and structures. Leukocyte common antigen (LCA) and thyroglobulin were negative on targeted structures, however, and with clear cross-reactivity on non-targeted tissues (vascular wall, granulocytes). Conclusive results were obtained when using polyclonal antibodies with dilution adjusted to laboratory practice, while application of ready-to-use (RTU) kits with pre-diluted antibodies or monoclonal antibodies often showed conflicting or inconclusive results.

Bisphenols emerging in Norwegian and Czech aquatic environments show transthyretin binding potency and other less-studied endocrine-disrupting activities.

Bisphenols are increasingly recognized as environmental pollutants with endocrine-disrupting potential. Nonetheless, the study of environmental occurrence and some endocrine-disrupting activities of some bisphenols came widely into focus of research only recently. The aims of the present study were to: 1) determine the predominant bisphenols in Norwegian sewage sludge and sediment and in Czech surface waters, and 2) characterize the binding of bisphenols to a transport protein transthyretin (TTR) and their (anti-)thyroid, (anti-)progestagenic, and (anti-)androgenic activities. High-performance liquid chromatography with atmospheric pressure chemical ionization or photoionization coupled with high resolution mass spectrometry (HPLC-APCI/APPI-HRMS) and Chemically Activated LUciferase gene eXpression (CALUX) in vitro reporter gene bioassays were used to detect the target compounds and to determine endocrine-disrupting activities, respectively. Bisphenol A (BPA), 4,4'-bisphenol F (BPF), bisphenol S (BPS), and bisphenol E (BPE) were the most frequently found compounds in municipal sewage sludge. Furthermore, bisphenol TMC (BPTMC) and bisphenol AF (BPAF) frequently occurred in sediment and surface waters, respectively. BPA was the major contributor to Ʃ of bisphenols in Norwegian sewage sludge with exception of one sample where BPF predominated. We also monitored a few bisphenols in sediment but only BPTMC was found. BPA, BPAF and BPF were the dominant bisphenols in Czech surface waters. Some bisphenols have shown TTR binding potency (BPAF = BPF > BPA = BPE) and some have displayed the following endocrine-disrupting activities: anti-thyroid (BPAF), anti-progestagenic (BPTMC > BPA = BPAF), and anti-androgenic (BPAF > BPE > BPA > BPTMC > BPF > BPS). It is noteworthy that BPAF exhibited stronger or similarly potent endocrine-disrupting activities compared to BPA. Our results provide new insights into these less-studied endocrine-disrupting activities of environmentally relevant bisphenols and may be useful in prioritizing those compounds that deserve further attention in environmental monitoring and eco-toxicological research.

Chronic simultaneous exposure of common carp (Cyprinus carpio) from embryonic to juvenile stage to drospirenone and gestodene at low ng/L level caused intersex.

Synthetic progestins are emerging contaminants of the aquatic environment with endocrine disrupting potential. The main aim of the present study was to investigate the effects of the synthetic progestins gestodene, and drospirenone on sex differentiation in common carp (Cyprinus carpio) by histological analysis. To gain insights into the mechanisms behind the observations from the in vivo experiment on sex differentiation, we analyzed expression of genes involved in hypothalamus-pituitary-gonad (HPG) and hypothalamus-pituitary-thyroid (HPT) axes, histology of hepatopancreas, and in vitro bioassays. Carp were continuously exposed to concentrations of 2 ng/L of single progestins (gestodene or drospirenone) or to their mixture at concentration 2 ng/L of each. The exposure started 24 h after fertilization of eggs and concluded 160 days post-hatching. Our results showed that exposure of common carp to a binary mixture of drospirenone and gestodene caused increased incidence of intersex (32%) when compared to clean water and solvent control groups (both 3%). Intersex most probably was induced by a combination of multiple modes of action of the studied substances, namely anti-gonadotropic activity, interference with androgen receptor, and potentially also with HPT axis or estrogen receptor.

Oxidative stress induced by fluoroquinolone enrofloxacin in zebrafish (Danio rerio) can be ameliorated after a prolonged exposure.

The purpose of our experiment was to evaluate the effect of enrofloxacin on biotransformation, oxidative stress and mRNA expression of related genes in fish as a non-target organisms. Zebrafish (Danio rerio) juveniles were treated with enrofloxacin at concentrations of 5, 10 and 500 µg/L for 14 days. A three-day-long test caused changes of catalytic activity of glutathione peroxidase and glutathione-S-transferase. Moreover, lipid peroxidation was observed at the highest concentration. No significant changes either in catalytic activity of antioxidant enzymes or elevated lipid peroxidation were observed from sampling day 7 on. mRNA expression of genes encoding antioxidant enzymes was also not affected by enrofloxacin after a 14-day exposure. This suggests the ability of D. rerio juveniles to adapt to enrofloxacin in a short time period. Moreover, enrofloxacin was not shown to affect collagen, cathepsin K, optic atrophy 1 and pyruvate kinase L/R mRNA expression in this study.

Synthetic progestin etonogestrel negatively affects mating behavior and reproduction in Endler's guppies (Poecilia wingei).

High rates of progestins consumption in the form of active ingredients in women's oral contraceptives and other hormonal preparations may lead to their increased concentrations in aquatic environments and subsequent harmful effect on fish reproduction. The objective of the present study was to assess the effect of etonogestrel, a third-generation synthetic progestin, on the reproductive behavior, fertility, gonads histology, and secondary sexual characteristics of male and female Endler's guppies (Poecilia wingei). Fish were subjected for 34 days to two concentrations of etonogestrel, including one possibly environmentally relevant (3.2 ng L-1) and one sublethal (320 ng L-1) concentration. A mating behavior study was subsequently conducted and revealed that the treatment with etonogestrel significantly reduced mating frequency in the exposed fish compared to controls. All the exposed females were unable to reproduce. In addition, female fish exposed to the highest level of etonogestrel were masculinized, as their anal fins and body coloration showed patterns similar to those of male fish. Etonogestrel-exposed females also had fewer developed oocytes. In conclusion, the low etonogestrel concentration (3.2 ng L-1) led to a reduction of mating activity in males without effect on their reproductive success, but it completely inhibited reproduction in females. Exposure to etonogestrel clearly has more severe consequences for females than males.

Effect of polycyclic musk compounds on aquatic organisms: A critical literature review supplemented by own data.

Synthetic musk compounds are extensively used in personal care and cosmetic products around the world. Because they are not completely removed in sewage treatment plants, they eventually end up in aquatic environments. The aim of this review was to summarize published information on effects of polycyclic musks on aquatic organisms and to discuss whether the experimental design of toxicological studies involving these substances could influence the results obtained. With the exception of one study run in a flow-through system, all published toxicological studies on synthetic polycyclic musks have been conducted in semi-static or even static systems. Based upon data in the literature and our own results, we conclude that in toxicological tests with semi-static set-ups, concentrations of polycyclic musks decrease with time between bath exchanges, and, as a result, tested organisms are not being exposed to stable concentrations but rather to concentration pulses. The duration and character of these pulses are influenced mainly by aeration of experimental baths, as polycyclic musks have a tendency to volatilize from water baths. Under semi-static conditions, tested organisms may be subjected to lower concentration of the tested substance for relatively long periods. Those levels may even fall below the limits of quantification. During these periods, some level of detoxification and/or elimination (depuration) of the toxicant may reduce toxic effect of the previous exposures. Consequently, toxicity of polycyclic musk substances for aquatic organisms obtained under these conditions may be underestimated. Based upon existing data in the literature, therefore, it is very difficult to correctly estimate risk of polycyclic musks to aquatic organisms.

Do progestins contribute to (anti-)androgenic activities in aquatic environments?

Unknown compounds with (anti-)androgenic activities enter the aquatic environment via municipal wastewater treatment plants (WWTPs). Progestins are well-known environmental contaminants capable of interfering with androgen receptor (AR) signaling pathway. The aim of the present study was to determine if 15 selected progestins have potential to contribute to (anti-)androgenic activities in municipal wastewaters and the respective recipient surface waters. AR-specific Chemically Activated LUciferase gene eXpression bioassay in agonistic (AR-CALUX) and antagonistic (anti-AR-CALUX) modes and liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) methods were used to assess (anti-)androgenic activity and to detect the target compounds, respectively. The contribution of progestins to (anti-)androgenic activities was evaluated by means of a biologically and chemically derived toxicity equivalent approach. Androgenic (0.08-59 ng/L dihydrotestosterone equivalents - DHT EQs) and anti-androgenic (2.4-26 µg/L flutamide equivalents - FLU EQs) activities and progestins (0.19-75 ng/L) were detected in selected aquatic environments. Progestins displayed androgenic potencies (0.01-0.22 fold of dihydrotestosterone) and strong anti-androgenic potencies (9-62 fold of flutamide). Although they accounted to some extent for androgenic (0.3-29%) and anti-androgenic (4.6-27%) activities in influents, the progestins' contribution to (anti-)androgenic activities was negligible (≤2.1%) in effluents and surface waters. We also tested joint effect of equimolar mixtures of target compounds and the results indicate that compounds interact in an additive manner. Even if progestins possess relatively strong (anti-)androgenic activities, when considering their low concentrations (sub-ng/L to ng/L) it seems unlikely that they would be the drivers of (anti-)androgenic effects in Czech aquatic environments.

Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.

Determination of progestogens in surface and waste water using SPE extraction and LC-APCI/APPI-HRPS.

The aim of this study was to develop a reliable analytical method for the measurement of 17 selected progestogens in waste water and surface water. Automated whole water solid phase extraction (SPE) was used for sample concentration. Liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric pressure photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) was applied for the analyses. The whole-method recoveries ranged from 60% to 140% for all analytes at two different spike levels (5 and 50ng/L) in the studied matrices. The method is very sensitive with LOQs ranging from 0.02 to 0.87ng/L. The developed method was used for the determination of progestogens in real samples of waste water from three waste water treatment plants (WWTPs) and in surface water from the corresponding recipients. Progesterone was detected in all samples with concentrations in the range of 0.82 to 1.1ng/L in surface water and 0.11 to 110ng/L in waste water samples. Three synthetic progestogens, namely, megestrol acetate, medroxyprogesterone acetate, and dienogest, were detected most frequently in effluents; therefore, further attention should be paid to the monitoring of these compounds. To the best of our knowledge, this study is the first to present analysis of altrenogest, etonogestrel, dienogest, nomegestrol acetate and ulipristal acetate in waste water and surface water using a solid-phase extraction method.

Effect of cadmium on uptake of iron, zinc and copper and mRNA expression of metallothioneins in HepG2 cells in vitro.

The intake of cadmium contaminated fish was mimicked by incubating human hepatoblastoma cells (Cell line HepG2) with a combination of different levels of cadmium (0-5µM) plus the n-3 fatty acids docosahexaenoic acid and eicosapentaenoic acid, which are typical for fish. Uptake of cadmium, iron, copper and zinc was measured by ICP-MS. In addition mRNA expression of two metallothioneins (mt1 g and mt1 m) was evaluated by real-time PCR. The obtained data shows that the presence of cadmium increases the uptake of iron and zinc into the HepG2 cells while the uptake of copper remains unaffected. The presence of the chosen fatty acids did not affect the uptake of either cadmium or iron, zinc and copper. The presence of already 1µM cadmium increased the mRNA expression of mt1 g and mt1 m significantly, while the fatty acids did not interfere with the effect of cadmium.

Comparison of passive sampling and biota for monitoring of tonalide in aquatic environment.

Synthetic musk compounds are extensively used in personal care and cosmetic products all over the world. Afterwards, they are discharged into the environment mainly because they are not completely removed in wastewater treatment plants. The aim of this study was to investigate if a passive sampler is applicable for the monitoring of tonalide, a polycyclic musk compound, in the aquatic environment and to compare the levels of tonalide in pesticide-polar organic chemical integrative sampler (POCIS) and biota. For this purpose, four sampling localities on the three biggest rivers in the Czech Republic were selected. Tonalide was determined in POCIS at all sampling sites in the concentration ranging from 9 ng/POCIS (Labe River, Hradec Králové) to 25 ng/POCIS (Morava River, Blatec). The locality with the most frequent occurrence of tonalide in biota samples was the Morava River which well corresponded with the highest tonalide concentration in POCIS among sampling sites. The highest number of positive tonalide detections among all studied biota samples was found in fish plasma. To the best of our knowledge, this is the first evidence that tonalide bioaccumulates in fish blood. Tonalide levels were below the limit of quantification in benthos samples at all sampling sites.

Effect of the human therapeutic drug diltiazem on the haematological parameters, histology and selected enzymatic activities of rainbow trout Oncorhynchus mykiss.

Diltiazem is a pharmaceutical belonging to a group of calcium channel blockers (CCB) that is widely used in the treatment of angina pectoris and hypertension. The objective of the present study was to assess the effect of diltiazem on rainbow trout (Oncorhynchus mykiss). Juvenile trout were exposed for 21 and 42 days to three nominal concentrations of diltiazem: 0.03 µg L(-1) (environmentally relevant concentration), 3 µg L(-1), and 30 µg L(-1) (sub-lethal concentrations). The number of mature neutrophilic granulocytes was significantly increased by 450 and 400% in fish exposed to 3 µg L(-1) and 30 µg L(-1) diltiazem compared to the control, respectively. Antioxidant enzyme activity was affected in liver and gills of fish exposed to all tested concentrations of diltiazem but the changes were mostly transient and not concentration dependent. Creatine kinase activity was markedly increased (ranging from 520 to 845%) at all tested diltiazem concentrations at the end of the exposure indicating muscle and/or kidney damage. The highest concentration was associated with histological changes in heart, liver, and kidney. These alterations can be attributed to the effects of diltiazem on the cardiovascular system, similar to those observed in the human body, as well as to its metabolism. At the environmentally relevant concentration, diltiazem was found to induce some alterations in the blood, gills, and liver of fish, indicating its potential for adverse effects on non-target organisms in the aquatic environment.