Quantitative assessment of estrogenic activity in the water environment of Korea by the E-SCREEN assay

In this study, the E-SCREEN assay was optimized and validated for the sensitive quantitative determination of the total estrogenicity in river samples. River water and sediment samples were collected and analyzed with the E-SCREEN. River water (10 l) was extracted using combined solid-phase extraction in static adsorption mode with Soxhlet extraction. Estrogenic pollutants adsorbed to the XAD-4 resin were recovered with 98.24 +/- 5.90% efficiency by elution with ethyl acetate and dichloromethane (1:9). The detection limit by 17beta-estradiol equivalent concentration (EEQ) of the E-SCREEN assay was 8.03 pg EEQ/l. Among the water samples, the estrogenic activity was observed to be higher downstream of the Kumho river (7.43 ng EEQ/l) and upstream of Kum river (2.05 ng EEQ/l) than in other samples. More than 3 mg of equivalent sediment samples from the Kumho river, Kum river and Miho stream showed partial agonistic effects, and the Mankyung river showed a partial agonistic effect with only 1.5 mg of sediment. The highest value of RPE was 83.34 downstream of the Kumho river, and the lowest value of RPE was 6.52 downstream of the Miho stream. Full estrogen agonistic activities were observed downstream of the Kumho river and upstream of the Kum river. The partial agonistic activity was observed in upstream of the Kumho river, downstream of the Mankyung river, and upstream of the Miho stream, and no agonistic action was observed downstream of the Kum river or Miho stream, or upstream of the Mankyung river. The total estrogenic activity in the river water and sediment samples was between 0.50 pg/L and 7.4 ng/L, 3.39 pg/g and 10.70 pg/g.     

Removal of estrogenic activities of 17beta-estradiol and ethinylestradiol by ligninolytic enzymes from white rot fungi

We investigated whether manganese peroxidase (MnP) and the laccase-mediator system with 1-hydroxybenzotriazole (HBT) as mediator can remove the estrogenic activities of the steroidal hormones 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)). Using the yeast two-hybrid assay system, we confirmed that the estrogenic activities of E(2) and EE(2) are much higher than those of bisphenol A and nonylphenol. Greater than 80% of the estrogenic activities of E(2) and EE(2) were removed following 1-h treatment with MnP or the laccase-HBT system; extending the treatment time to 8h removed the remaining estrogenic activity of both steroidal hormones. HPLC analysis demonstrated that E(2) and EE(2) had disappeared almost completely in the reaction mixture after a 1-h treatment. These results strongly suggest that these ligninolytic enzymes are effective in removing the estrogenic activities of E(2) and EE(2).     

Contribution of known endocrine disrupting substances to the estrogenic activity in Tama River water samples from Japan using instrumental analysis and in vitro reporter gene assay

To quantitatively characterize the substances contributing to estrogenic activity in river water, in vitro bioassay using MVLN cells and instrumental analysis using liquid chromatograph–mass spectrometer (LC/MS) or liquid chromatograph–tandem mass spectrometer (LC/MS/MS) were applied to river water extracts taken from various locations in the Tama River, Japan. Tama River water samples were extracted using solid phase extraction and the crude extracts were fractionated by high-performance liquid chromatography (HPLC) into 10 fractions. The sixth fraction contained nonylphenol (NP) and octylphenol (OP) at concentrations in the range of 51.6–147 and 6.9–81.9 ng/L, respectively (concentrations corresponding to the original sample volumes). No estrogenic activity, expressed as 17β-estradiol equivalents (E2-EQ_B), however, was observed in this fraction (<0.6 ng-E2eq/L). Instrumentally determined estrogenic activity (E2-EQ_C), which is the concentrations of NP and OP multiplied by their corresponding relative potency, was below the detection limit of the MVLN cell bioassay. Estrogenic activities were detected only in HPLC fraction nos. 7, 8 and 9. Estrone (E1), estradiol (E2) and bisphenol A (BPA) were detected in these fractions. Estriol (E3) and ethynylestradiol (EE2) were not detected (<0.2 ng/L) in these fractions. The calculated E2-EQ_C for BPA was below the detection limit of bioassay. The E2-EQ_C for E1 and E2 were on the same order as the estrogenic activity determined by the bioassay (E2-EQ_B). The ratios of E2-EQ_C and E2-EQ_B for E1 and E2 in the three factions collectively (nos. 7–9) were 0.49–0.97 and 0.29–1.12, respectively. Above results indicated that the major causal substances to the estrogenic activity in the Tama River were E1 and E2.     

Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast assay

A scheme of bioassay-directed analysis has been developed which combines a yeast assay screening for estrogenic activity with a liquid chromatographic-mass spectrometric (LC-MS/MS) chemical analysis, chromatographic fractionation, solid phase extraction and freeze-drying. The test scheme was applied on effluent samples collected from a municipal sewage treatment plant. The aim was to determine the substances responsible for main portion of the estrogenic activity in the samples and to compare the efficiency of different procedures for isolation and concentration of estogenicity. LC-MS/MS analyses were used for the quantification of 17beta-estradiol, estrone, estriol and 17alpha-ethinylestradiol, and the measured concentrations compared with the activities found in the yeast assay. Following conversion of the concentrations measured by LC-MS/MS to 17beta-estradiol equivalents it was concluded that freeze-drying, solid phase extraction and the chemical analysis gave comparable activities. Since estrone was the major estrogen in the effluent, this estrogen was also the major contributor to the estrogenic activity in the effluent. The estrogenic activity was equivalent to 4-7 ng/L of 17beta-estradiol. The yeast assay results from the tests of the chromatographic fractions showed that the major activity resides in the fraction where estrone, 17beta-estradiol and 17alpha-ethinylestradiol eluted. The activity of this fraction was substantially higher than the activity of the original wastewater sample. The reason for this could in part be explained by an inhibition of activity occurring in the original water sample.     

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.     

Monitoring of dioxin-like, estrogenic and anti-androgenic activities in sediments of the Bizerta lagoon (Tunisia) by means of in vitro cell-based bioassays: contribution of low concentrations of polynuclear aromatic hydrocarbons (PAHs)

We used an array of in vitro cell-based bioassays to assess dioxin-like, estrogenic and (anti-)androgenic activities in organic extracts of sediments from the Bizerta lagoon, one of the largest Tunisian lagoons subjected to various anthropogenic and industrial pressures. The sediments were sampled both in winter and summer 2006 in 6 stations differently impacted and in one reference station located in the seawards entrance of Ghar el Melh lagoon. Chemical analyses of the 16 priority PAHs showed that the sediments were low to moderately contaminated (2-537 ng/g dry weight). By using the estrogen- (MELN) and androgen-responsive (MDA-kb2) reporter cell lines, significant estrogenic and anti-androgenic activities were detected only in the Menzel Bourguiba (MB) site, the most contaminated site, both in winter and summer. By using 7-ethoxyresorufin-O-deethylase (EROD) induction in the fish PLHC-1 cell line after both 4 and 24 h of cell exposure, dioxin-like activities were detected in all analysed samples. Dioxin-like activities were higher after 4 h exposure, and varied according to the sites and the sampling season. While highly significant correlation was observed between bioassay- and chemical analyses-derived toxic equivalents (TEQs), PAHs accounted for only a small part (up to 4%) of the detected biological activities, suggesting that other readily metabolised EROD-inducing compounds were present. This study argues for the use of short time exposure to assess biological TEQs in low contaminated samples and provides new induction equivalent factors (IEF(4h)) for 16 PAHs in the PLHC-1 cell line. Finally, our results stress the need to further characterise the nature of organic chemical contamination as well as its long-term impacts on aquatic wildlife in the Bizerta lagoon.     

Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents

A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.     

超声空化增强川芎嗪对脑缺血再灌注损伤保护作用机制的研究

川芎的生物活性成分(川芎嗪)已广泛应用于治疗心脑血管疾病。基于谷氨酸诱导PC12细胞损伤建立脑缺血再灌注损伤的细胞模型,探讨超声增强川芎嗪对谷氨酸损伤PC12细胞的保护作用机制。研究结果表明,超声能进一步加强川芎嗪对细胞的保护,其主要作用机制为:(1)抑制氧化应激和细胞凋亡相关的Bcl-2蛋白和Bax蛋白的变化从而达到抗凋亡的效果;(2)降低炎症因子(TNF-α和IL-8)的表达,减轻炎症反应损伤;(3)适当的声压可以增强川芎嗪对谷氨酸损伤PC12细胞的保护作用,但过高的声压会引起细胞损伤,导致细胞凋亡。本文的工作表明超声能够增强川芎嗪对脑缺血再灌注损伤的保护作用,为临床脑缺血再灌注损伤的治疗提供了新治疗策略。     

Removal of organophosphate pesticides from wastewater by supercritical carbon dioxide extraction

Organophosphate pesticides including fenitrothion chlorpyrifos, diazinon, methamidophos, edifenphos, mevinphos, fenthion, and acephate present in agro-wastewater can be effectively removed by supercritical carbon dioxide (SC-CO2) extraction. Near quantitative removal of the pesticides from the aqueous solution can be achieved by SC-CO2 at 90 degrees C and 325 atm for 20 min of static extraction followed by 40 min of dynamic extraction. The extracted pesticides were collected in a small amount of Fenton's solution. The pesticides in Fenton's solution were degraded completely within an hour after the collection. A combination of SC-CO2 extraction and subsequent degradation by Fenton's reagent may provide an alternative water purification strategy for treating organophosphate pesticides in agro-wastewater.     

库区边坡勘察与稳定性分析

介绍了边坡工程地质条件，分析了边坡变形的特征及成因，阐述了边坡稳定性的计算方法，并对其计算结果作了分析，对边坡的变形、检测等得出一些结论。     

Presence of carbamate pesticides in environmental waters from the northwest of Mexico: determination by liquid chromatography

A study on the presence of carbamate pesticides, namely aldicarb, aldicarb sulfoxide, baygon, benthiocarb, carbofuran, 3-hydroxycarbofuran, carbaryl, desmedipham, methiocarb, methomyl, thiodicarb, oxamyl, and propham was made in ground and surface waters from an agricultural zone of the Yaqui Valley located in northwest Mexico. Trace determinations were made by liquid chromatography (LC) with post-column fluorescence detection (EPA method 531.1) or LC-diode array UV detection coupled on-line to a solid-phase extraction (SPE) system and using a 13 x 4.6 mm i.d. precolumn and a 150 x 4.6 mm i.d. analytical column, both packed with a C18 silica. Results indicated that the level of contamination with methiocarb was 5.4 micrograms/L in a groundwater sample and that for 3-hydroxycarbofuran was 18 micrograms/L in a surface water sample. This study provides the basis for a future monitoring program.     

Assessment of river contamination by estrogenic compounds in Paris area (France)

Wastewater treatment plants (WWTPs) receive a large spectrum of endocrine disrupting compounds (EDCs) that are partially eliminated during treatment processes and discharged into rivers. Given the lack of information in France about river contamination by EDCs, we chose to examine estrogenic potential of WWTP influents, effluents and receiving waters in Paris and its suburbs. Water samples were analyzed using gas chromatography coupled with mass spectrometry for quantifying natural and synthetic estrogens combined with an in vitro estrogenicity bioassay associated to a high pressure liquid chromatography fractionation. The four estrogens investigated, Estrone (E1), 17beta-Estradiol (E2), Estriol (E3) and 17alpha-Ethinylestradiol (EE2) were found in all WWTP and river samples at concentrations ranging from 2.7 to 17.6 ng/l and 1.0 to 3.2 ng/l, respectively. The synthetic estrogen EE2 seems more resistant to biodegradation in WWTPs and thus accounted for 35-50% of the estimated estrogenic activity in rivers. However, fractionation of samples and differences between concentrations of E1, E2, E3 and EE2 and the estrogenic activity measured by the in vitro bioassay suggested a complexity of mechanisms underlying the biological response that could not be attributed only to the investigated molecules.     

Factors involved in the dynamics of pesticides in soils: the effect of temperature and period of contact on leachability and adsorption of pesticides by soils

The effect of soil temperature on Ieachability and adsorption of pyrolan, γ‐HCH, α‐endosulphan, β‐endosulphan and dieldrin by Gezira soil from the Sudan, was studied. It was found that even when subjected to high temperatures (45°C) and prolonged leaching (60+ days), dieldrin and endosulphan isomers did not leach below 17 cm depth, but pyrolan and γ‐HCH exhibited leaching. In general, the leaching of pyrolan was greater than that of γ‐HCH. With the exception of pyrolan, the pesticide adsorption decreased progressively with temperature. Pyrolan adsorption was only slightly affected by temperature as most of the compound was either degraded or recovered from the leachate. Unaccountable losses of all pesticides increased with temperature. The effect of different periods of contact and temperature on adsorption of pesticides by a sandy clay loam from Northern England was also investigated.     

Effects of chlorine on the decrease of estrogenic chemicals

The effects of chlorination on the elimination of three estrogenic chemicals such as 17beta-estradiol, nonylphenol and bis-phenol A were investigated using yeast two-hybrid assay (YTA), estrogen receptor (ER) competition assay (ER-CA), and high-performance liquid chromatography/mass spectrometry (LC/MS). The results of YTA, ER-CA and the analysis of LC/MS indicated that the estrogenic activity of the above-mentioned three endocrine disruptors were significantly reduced as a result of chlorination. The decrease in estrogenic activity paralleled a decrease in estrogenic chemicals under the influence of free chlorine. One common characteristic of estrogenic chemicals is the presence of a phenolic ring. Considering that a phenolic ring is likely to undergo some sort of transformation in an aqueous chlorination solution, the above-mentioned results may be applied to the rest of the estrogenic chemicals in natural waters.     

Determination of chlorinated pesticides in water by SPME/GC

Solid-phase microextraction (SPME) followed by gas chromatography (GC) coupled with an electron capture detector has been applied for the analysis of chlorinated pesticides in water. Molecule adsorption on 100 microm polydimethylsiloxane (PDMS) fibers was activated by immersion of the whole fiber-sample system in an ultrasonic bath. The good reproducibility, low detection limits and wide linear ranges obtained encourage the use of this technique in water control.     

The impact of climate change on aquatic risk from agricultural pesticides in the US

We investigate how climate change may affect the acute and chronic toxicity risk to aquatic species from agricultural pesticides in 32 States of the US. We combine climate change projections from the Canadian and Hadley climate model, statistically estimated relationships between pesticide applications and climate and weather variables, and the environmental risk indicator REXTOX developed by the OECD. On average, we find that climate change is likely to increase the toxicity risk to aquatic species by 47% because of increased applications of agricultural pesticides. Daphnia and fish are more affected than algae. Across eight broad crop groups, pesticides used on pome and stone fruits and on fruiting vegetables contribute the most to aquatic risk. Within the 32 US States examined, more than 90% of the pesticide pollution impacts induced by climate change on the aquatic environment are caused by only 13 States near to the coast.     

Selective removal of estrogenic compounds by molecular imprinted polymer (MIP)

A molecular imprinted polymer (MIP) was synthesized and used for selective removal of estrogenic compounds. The study on the mass balance of template as well as template leakage revealed that almost all the template could be extracted out of the polymer and no template leakage could be detected in aqueous solution. In acetonitrile, MIP could adsorb more E2 than non-template imprinted polymer (NIP) by more than 3.5 times. In aqueous solution, MIP showed reduced selectivity for E2 with a 10% difference in adsorption capacity between MIP and NIP. With an initial E1, E2, EE2 and BPA concentration of 4muM in aqueous solution and adsorbent concentrations of 0.01-0.5mg/mL, the maximum adsorption capacities of MIP for E1, E2, EE2 and BPA were 92.8, 95.8, 115.4 and 57.4mumol/g polymer, respectively. According to the adsorption isotherms for E1, E2, EE2 and BPA, a physical adsorption model containing three types of binding sites, namely, specific binding site, semi-specific binding site and non-specific binding site, was proposed to interpret the adsorption performance of MIP.     

城市照明色光对居民室内活动的干扰阈值

城市照明的发展与LED的广泛使用致使居住区室外光环境日益明亮,且常见彩色光,居民的室内休闲娱乐活动所受到的影响也日见显著。从居民的主观感受出发,通过主观评价实验,得出正常室内照明情况下城市照明色光对居民看电视的干扰阈值,包括表征居民视觉感知的"明度阈值"和表征光源实际情况的"亮度阈值"。讨论了城市照明色光对居民室内活动干扰的季节差异、性别差异和色调差异,并对阈值结论的应用性进行了讨论。