Identification of new endocrine disruptive transthyretin ligands in polluted waters using pull-down assay coupled to non-target mass spectrometry.

Surface and treated wastewater are contaminated with highly complex mixtures of micropollutants, which may cause numerous adverse effects, often mediated by endocrine disruption. However, there is limited knowledge regarding some important modes of action, such as interference with thyroid hormone (TH) regulation, and the compounds driving these effects. This study describes an effective approach for the identification of compounds with the potential to bind to transthyretin (TTR; protein distributing TH to target tissues), based on their specific separation in a pull-down assay followed by non-target analysis (NTA). The method was optimized with known TTR ligands and applied to complex water samples. The specific separation of TTR ligands provided a substantial reduction of chromatographic features from the original samples. The applied NTA workflow resulted in the identification of 34 structures. Twelve compounds with available standards were quantified in the original extracts and their TH-displacement potency was confirmed. Eleven compounds were discovered as TTR binders for the first time and linear alkylbenzene sulfonates (LAS) were highlighted as contaminants of concern. Pull-down assay combined with NTA proved to be a well-functioning approach for the identification of unknown bioactive compounds in complex mixtures with great application potential across various biological targets and environmental compartments.

Bioaccumulation as a method of removing psychoactive compounds from wastewater using aquatic plants.

Since WWTPs are not able to eliminate all psychoactive pharmaceuticals, these compounds become a part of the aquatic ecosystem. Our results indicate that compounds such as codeine or citalopram are eliminated with low efficiency (<38%), and compounds such as venlafaxine, oxazepam, or tramadol even with almost no efficiency. Lower elimination efficiency may be caused by the accumulation of these compounds in the wastewater treatment process. This study is focused on the possibility to remove problematic psychoactive compounds using aquatic plants. HPLC-MS analysis of the leaf extract obtained from studied plants showed that the amount of accumulated methamphetamine was highest in Pistia stratiotes and lower in the leaves of Limnophila sessiliflora and Cabomba caroliniana. However, tramadol and venlafaxine were accumulated considerably only in Cabomba caroliniana. Our study demonstrates that especially these three compounds - tramadol, venlafaxine, and methamphetamine, are accumulated in aquatic plants and can be removed from the aquatic environment. In our study was also observed that helophytic aquatic plants show a higher ability to remove psychoactive compounds from wastewater. Iris pseudacorus showed the best results in selected pharmaceuticals removal with no bioaccumulation effect in leaves or roots.

Ultrafast laser diode thermal desorption method for analysis of representative pharmaceuticals in soil leachate samples.

We developed and evaluated a novel analytical method combining ambient ionization technique - laser diode thermal desorption with chemical ionization (LDTD-APCI) and tandem mass spectrometry detection. The LDTD/APCI-MS/MS method was developed for determination of representative pharmaceuticals from different classes (carbamazepine, sulfamethoxazole, irbesartan, fexofenadine) in leachate samples from soil sorption experimentation. We then optimized laser pattern, laser energy and spiked sample volume, which are crucial parameters for this LDTD/APCI-MS/MS method. We further identified utility of a chelating agent (Na2-EDTA) to obtain the highest achievable and reproducible signal of target analytes. Achieved method performance parameters (LODs, LOQs, trueness and precision) were comparable with those obtained from LC-MS/MS. However, application of this novel LDTD/APCI-MS/MS method reduced analysis time by two orders of magnitude (to 12 s), compared to more conventional LC-MS/MS approaches, without use of organic solvents. We expect this novel method will reduce costs and increase throughput for future analyses of pharmaceuticals in the environment while advancing a timely principle of green chemistry.

Behaviour and cardiac response to stress in signal crayfish exposed to environmental concentrations of tramadol.

Evidence of the ecological and biological impact of pharmaceuticals in surface waters on aquatic organisms is increasing. Tramadol is a synthetic opioid analgesic used to treat chronic and acute pain. To investigate its long-term effects at environmentally relevant levels, we evaluated heart rate (HR) and locomotion of signal crayfish Pacifastacus leniusculus during a 21-day exposure to 1 µg L-1 tramadol followed by 14 days depuration. Locomotion and HR were recorded over a period 30 min before and 30 min after exposure to physiological fluids of an injured conspecific, a natural stressor, four times during the tramadol exposure and four times during depuration. A significant increase in HR following stress induction was found in the majority of tramadol-exposed and control crayfish, as well as significant group-specific HR changes between both groups. Locomotor activity during tramadol treatment differed from that during depuration, in general showing less time spent in locomotion and lower distance moved. The tramadol exposed crayfish exhibited higher velocity during depuration than during the exposure period. Results may suggest a potential shift in prey-predator relationships.

Environmentally relevant concentrations of tramadol and citalopram alter behaviour of an aquatic invertebrate.

Environmental pollution by pharmaceutically active compounds, used in quantities similar to those of pesticides and other organic micropollutants, is increasingly recognized as a major threat to the aquatic environment. These compounds are only partly removed from wastewaters and, despite their low concentrations, directly and indirectly affect behaviour of freshwater organisms in natural habitats. The aim of this study was to behaviourally assess the effects of an opioid painkiller (tramadol) and antidepressant drug (citalopram) on behaviour patterns of a clonal model species, marbled crayfish. Animals exposed to environmentally relevant concentrations of both tested compounds (∼1 µg l-1) exhibited significantly lower velocity and shorter distance moved than controls. Crayfish exposed to tramadol spent more time in shelters. Results were obtained by a simple and rapid method recommended as suitable for assessment of behaviour in aquatic organisms exposed to single pollutants and combinations.

Quantification of adenosine triphosphate, adenosine diphosphate, and creatine phosphate in sterlet spermatozoa during maturation.

Sturgeon spermatozoa maturation during their passage through the kidney is a prerequisite for initiation of motility. Samples of sterlet () testicular sperm (TS) were matured in vitro by incubation in seminal fluid (SF) or in SF supplemented with carbonyl cyanide -chlorophenyl hydrazone (CCCP; a respiration uncoupling agent). Sperm was diluted in activation medium (AM) containing 10 m Tris-HCl buffer (pH 8.5) and 0.25% Pluronic, and spermatozoon motility was assessed. Samples were taken and fixed in 3 perchloric acid at 3 points in the incubation process. Quantification of ATP, ADP, and creatine phosphate (CrP) was conducted using liquid chromatography/high-resolution mass spectrometry. We observed a significant decrease in CrP during artificial maturation of TS in SF. In contrast, ATP and ADP were not significantly affected. Addition of CCCP to SF halted maturation and led to significantly lower CrP whereas ADP significantly increased and ATP was unaffected. Dilution of matured and immature TS with AM led to a significant decrease of ATP and CrP and an increase of ADP compared with their levels before dilution, although immature TS were not motile. Energy dependency of TS maturation in sturgeon was confirmed, which suggests that mitochondrial oxidative phosphorylation is needed for maturation of sturgeon TS.

The progestin levonorgestrel disrupts gonadotropin expression and sex steroid levels in pubertal roach (Rutilus rutilus).

The aim of the present study was to investigate the effects of the synthetic progestin levonorgestrel (LNG) on the reproductive endocrine system of a teleost fish, the roach (Rutilus rutilus). Pubertal roach were exposed for 28 days in a flow-through system to four concentrations of LNG (3, 31, 312, and 3124 ng/l). Both males and females treated with 3124 ng/l LNG exhibited the upregulated levels of vitellogenin and oestrogen receptor 1 mRNA in the liver. At the same concentration, LNG caused a significant upregulation of the mRNA expression of the gene encoding luteinising hormone ß-subunit (lhß) and the suppression of the mRNA expression of the gene encoding follicle-stimulating hormone ß-subunit (fshß) in the pituitary of both male and female roach. A lower LNG concentration (312 ng/l) suppressed mRNA expression of fshß in males only. Females treated with 3124 ng/l LNG exhibited significantly lower plasma 11-ketotestosterone (11-KT) and oestradiol (E2) concentrations, whereas their testosterone (T) level was higher compared with the control. Females exposed to 312 ng/l LNG presented significantly lower plasma E2 concentrations. Males exposed to ≥31 ng/l LNG exhibited significantly reduced 11-KT levels. As determined through a histological analysis, the ovaries of females were not affected by LNG exposure, whereas the testes of males exposed to 31 and 312 ng/l LNG exhibited a significantly higher percentage of spermatogonia B compared with the control. The results of the present study demonstrate that LNG disrupts the reproductive system of pubertal roach by affecting the pituitary gonadotropin expression and the sex steroid levels. This disruption was determined to occur in males after exposure to an environmentally relevant concentration (31 ng/l). Moreover, the highest tested concentration of LNG (3124 ng/l) exerted an oestrogenic effect on fish of both sexes.

A passive sampling method for detecting analgesics, psycholeptics, antidepressants and illicit drugs in aquatic environments in the Czech Republic.

The goal of this study was to assess the bioavailable concentrations of analgesics, psycholeptics, antidepressants and illicit drugs in the surface waters of the Czech Republic. All of the sampling sites are located within the most important water quality monitoring profiles at the Czech Hydrometeorological Institute. The total concentrations of the compounds ranged from 463 to 6,447 ng POCIS(-1) (Polar Organic Chemical Integrative Sampler). Carbamazepine (196-2,690 ng POCIS(-1)) and tramadol (160-2,250 ng POCIS(-1)) were the most abundant compounds at every site. The most polluted sites were those that received communal wastewater effluent and had a low dilution factor (ratio of wastewater effluent and river flow). The aqueous concentrations of the target compounds were estimated using sampling rate values obtained during a field calibration experiment. Patterns in the aqueous concentrations of the compounds (after back calculation from POCIS extracts) and the POCIS concentrations are different, possibly leading to discrepancies between the toxicity assessments conducted using POCIS extracts and those conducted using grab samples of water from the same location.

Estrogen-, androgen- and aryl hydrocarbon receptor mediated activities in passive and composite samples from municipal waste and surface waters.

Passive and composite sampling in combination with in vitro bioassays and identification and quantification of individual chemicals were applied to characterize pollution by compounds with several specific modes of action in urban area in the basin of two rivers, with 400,000 inhabitants and a variety of industrial activities. Two types of passive samplers, semipermeable membrane devices (SPMD) for hydrophobic contaminants and polar organic chemical integrative samplers (POCIS) for polar compounds such as pesticides and pharmaceuticals, were used to sample wastewater treatment plant (WWTP) influent and effluent as well as rivers upstream and downstream of the urban complex and the WWTP. Compounds with endocrine disruptive potency were detected in river water and WWTP influent and effluent. Year-round, monthly assessment of waste waters by bioassays documented estrogenic, androgenic and dioxin-like potency as well as cytotoxicity in influent waters of the WWTP and allowed characterization of seasonal variability of these biological potentials in waste waters. The WWTP effectively removed cytotoxic compounds, xenoestrogens and xenoandrogens. There was significant variability in treatment efficiency of dioxin-like potency. The study indicates that the WWTP, despite its up-to-date technology, can contribute endocrine disrupting compounds to the river. Riverine samples exhibited dioxin-like, antiestrogenic and antiandrogenic potencies. The study design enabled characterization of effects of the urban complex and the WWTP on the river. Concentrations of PAHs and contaminants and specific biological potencies sampled by POCIS decreased as a function of distance from the city.

Changes in concentrations of hydrophilic organic contaminants and of endocrine-disrupting potential downstream of small communities located adjacent to headwaters.

Endocrine-disruptive potential and concentrations of polar organic contaminants were measured in seven headwaters flowing through relatively unpolluted areas of the Czech Republic. Towns with Wastewater Treatment Plant (WWTP) discharges were the first known sources of anthropogenic pollution in the areas. River water was sampled several kilometers upstream (US) and several tens of meters downstream (DS) of the WWTP discharges, by use of Pesticide and Pharmaceutical Polar Organic Integrative Samplers (POCIS-Pest, POCIS-Pharm). Extracts of passive samplers were tested by use of a battery of in vitro bioassays to determine overall non-specific cytotoxicity, endocrine-disruptive (ED) potential and dioxin-like toxicity. The extracts were also used for quantification of polar organics. There was little toxicity to cells caused by most extracts of POCIS. Estrogenicity was detected in all types of samples even though US locations are considered to be background. At US locations, concentrations of estrogen equivalents (EEq) ranged from less than the detection limits (LOD) to 0.5 ng EEq/POCIS. Downstream concentrations of EEqs ranged from less than LOD to 4.8 ng EEq/POCIS. Concentrations of EEq in POCIS extracts from all DS locations were 1 to 14 times greater than those at US locations. Concentrations of EEq measured in extracts of POCIS-Pest and POCIS-Pharm were in a good agreement. Neither antiestrogenic nor anti/androgenic activities were detected. Concentrations of 2,3,7,8-TCDD equivalents (TEq(bio)) were detected in both types of POCIS at concentrations ranging from less than the LOD to 0.39 ng TEq(bio)/POCIS. Nearly all extracts of POCIS-Pharm contained greater concentrations of TEq(bio) activity than extracts of POCIS-Pest. Concentrations of pesticides and pharmaceuticals in extracts of POCIS were generally small at all sampling sites, but levels of some pharmaceuticals were significantly greater in both types of POCIS from DS locations. Chemical analyses along with the results of bioassays documented impacts of small towns with WWTPs on headwaters.

Effects of sulfur dioxide, hydrogen peroxide and sulfuric acid on the de novo synthesis of PCDD/F and PCB under model laboratory conditions.

In a laboratory model system consisting of fly ash from municipal waste incinerator, CuCl2 x 2H2O, NaCl and activated carbon in N2 + 10% O2 atmosphere, the de novo synthetic reactions of formation of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) were studied under laboratory conditions in the presence of sulfur dioxide, hydrogen peroxide, and sulfuric acid. It has been found that the formation of PCDD is suppressed by sulfur dioxide more efficiently than the formation of PCDF. A similar effect has also been observed in the presence of hydrogen peroxide. The formation of PCDF is strongly suppressed in the presence of sulfuric acid. On the basis of the experimental results and thermodynamic calculations, the following mechanisms are proposed and discussed: oxidative destruction of PCDD and PCDF oxygen rings, conversion of cupric chloride and possibly also cupric oxide into the non-reactive sulfate, and the Deacon oxychlorination processes catalyzed by cupric chloride.

Bioaccumulation of PCB congeners in marine benthic infauna.

Field concentrations of polychlorinated biphenyls (PCBs) were measured in sediment and nine marine soft bottom invertebrate species. Lipid- and organic carbon normalised biota-sediment accumulation factors (BSAFs) were determined for 29 nonplanar and 11 coplanar congeners. To investigate whether the bioaccumulation was in thermodynamic equilibrium with the sediment, the determined BSAFs were compared to theoretically calculated ones to obtain a BSAFdet./BSAFtheor-ratio. Large interspecific variations were found: one suspension feeding and one deposit feeding species of brittle stars (Amphiura filiformis and A. chiajei), and one predatory polychaete (Glycera rouxii) had ratios>1 for congeners with logKow>6.5. In these species there was also a linear relationship between logKow and BSAF, both for coplanar and for planar congeners but with lower values for coplanar ones. For other species the pattern was more scattered. Only the deposit feeding polychaete Melinna cristata had BSAFdet./BSAFtheor-ratios<1 for all congeners. Thus, the interspecific variations in bioaccumulation did not correlate with differences in feeding strategies, but may be caused by differences in biotransformation, and in age and size of the analysed specimens.

Differential accumulation of low-chlorinated (Delor 103) and high-chlorinated (Delor 106) biphenyls in human placental tissue and opposite effects on conversion of DHEA to E2.

OBJECTIVE: Polychlorinated biphenyls (PCBs) and related compounds elicit a diverse spectrum of toxic responses. Additionally, they are able to pass through the human placenta. The aim of the presented data was to compare the action of low-chlorinated (Delor 103) and (Delor 106) high-chlorinated biphenyls on placental steroidogenesis. METHODS: Explants of human placental tissue were used to test differences in PCBs accumulation and influence on placental steroidogenesis. Delor 103 or 106, were added daily for six days at a dose of 200 pg from day 0 to day 6 of culture. The media in the control and experimental groups were changed every day, and collected and frozen for steroid analysis by RIA. Determinations of PCBs of tissue and medium were analysed by GC/MS/MS. RESULTS: Delor 103 was found at a higher level in the tissue than Delor 106. The first day of exposure to Delor 103 had no effect on the conversion of dehydroepiandrosterone (DHEA) to estradiol (E2) while there was a 2-fold decrease in E2 secretion from days 3 to 6. Conversely, Delor 106 caused an immediate increase in E2 secretion, which was maintained at higher levels throughout the exposure period. CONCLUSION: Differences between the accumulation of lower chlorinated and higher chlorinated biphenyls in human placental tissue and in the properties of the congeners can have multiple effects that may intensify or counteract the effects on uterine contraction by PCBs.

Effect of reaction time on PCDD and PCDF formation by de novo synthetic reactions under oxygen deficient and rich atmosphere.

The effect of reaction time on formation of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) was studied under laboratory conditions in the system containing municipal waste incineration fly ash, activated carbon and copper chloride dihydrate at 300 degrees C in 99.999% N2 and N2 + 10% O2 atmosphere. The concentrations of tetra- to octa-chlorinated isomers as well as I-TEQ concentrations of toxic congeners are reported. The mechanism of PCDD and PCDF formation from chlorophenols and chlorinated biphenyls is discussed in the light of the time changes of PCDD/PCDF ratios.

Effects of oxygen on formation of PCB and PCDD/F on extracted fly ash in the presence of carbon and cupric salt.

The effect of oxygen-nitrogen atmosphere (N2 + 10%O2, N2 + 1%O2 and 99.999% N2) on the formation of PCB, PCDD and PCDF by the de novo synthetic reactions in the system consisting of extracted fly ash (from municipal waste incinerators--MWI), activated carbon, CuCl2 x 2H2O and NaCl at 340 degrees C was studied. The content of PCDD/F for systems with 10%O2, 1%O2 and 99.999% N2 was decreasing and corresponded to 17,304, 5544, and 1437 ng/sample. In all studied systems the isomer OCDD/F was prevailing. The content of PCBs in the same system was also decreasing from 1214 to 166 ng/g. Formation of nonortho PCB was relatively high compared to the system where only nitrogen was present. The possible mechanism of formation is outlined.

Dechlorination of polychlorinated biphenyls, dibenzo-p-dioxins and dibenzofurans on fly ash.

Dechlorination of commercial mixtures of polychlorinated biphenyls (PCB) as well as polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) on extracted and non-extracted fly ash obtained from municipal waste incinerator (MWI) was studied in closed systems under nitrogen atmosphere at temperatures of 260 degrees C and 340 degrees C. Decomposition results (given as the difference between PCB or PCDD/F molar amounts before and after the experiment (in %) due predominantly to dechlorination reactions) and detoxification data (expressed similarly but related to toxic PCB and PCDD/F congeners only and given in I-TEQ units) are reported. Detoxification of Delor 105/80T at 260 degrees C and 340 degrees C at a loading of 0.65 wt%, was 99.48% and 100%, respectively. The decomposition of Delor 103 at 340 degrees C and for the loading of 0.75 wt%, corresponded to 99.99%. The detoxification capability of PCDD/Fs on extracted and non-extracted fly ash for loading of 130 and 264 ng/0.4 g of fly ash at 340 degrees C made 96 and 98%, respectively.

The effect of oils on PAH, PCDD, PCDF, and PCB emissions from a spark engine fueled with leaded gasoline.

The effect of synthetic and mineral oils on the formation of polyaromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs) in emissions from a spark ignition engine was studied on a Skoda Favorit engine fueled with leaded gasoline. The test cycle simulated urban traffic conditions on a chassis dynamometer, in accordance with the ECC 83.00 test. The data for selected PAHs as well as PCDDs, PCDFs, and PCBs congener profiles are presented. PCDD/Fs emissions for an unused oil and the oil after 10000-km operation varied from 300 to 2000 fmol/m3, PCBs emissions from 75 to 178 pmol/m3, and PAHs emissions from 150 to 420 microg/m3. The content of PCBs in oils varied from 2 to 920 mg/kg.