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Feminisation of fish 4. Quantification of the estrogenicity of sewage effluent using in vitro assays and the TIE approach.The observations of feminisation of fish exposed to sewage effluent, indicating that the effluent contains estrogenic compounds, have led to attempts of quantifying the estrogenic activity of the effluent. Fractionation techniques followed by in vitro assays have been used to quantify the estrogenic activity of the sewage effluent. The fractions responsible for the estrogenic activities in the in vitro assays have in some cases further been subjected to chemical analyses leading to identification of the active chemicals (25) - a procedure called toxicology identification evaluation (TIE). The following chapter will summarise the results made on estrogenicity of sewage effluent with numerous in vitro assays and compare these with the actual concentrations measured in the sewage effluent where these have been made. Additional results on estrogen concentrations from chemical analysis of sewage effluent and surface water will be presented in chapter 5. The additive behaviour of the estrogenic activity of single substances in a mixture has been demonstrated and is the basis for quantitatively assessing the total contents of estrogenic substances in an environmental sample by the use of in vitro assays. The total estrogenicity is then compared to the magnitude of response elicited by 17ß-estradiol and described as estrogen equivalents (30). A number of cell based in vitro assays have been applied which uses cells transfected with an estrogen receptor controlled reporter gene (30;30;32). Among these is the YES assay using yeast cells transfected with the human estrogen receptor (ER) and the reporter gene b -galactosidase (25). Other assays use transfected human breast cancer cells transfected i.e. with luciferase in the ER-CALUX assay (32). The human breast cancer cells, of which different strains have been used, already contain the human estrogen receptor. The E-screen, in which proliferation of human breast cancer cells (MCF-7) as a response to estrogen is measured, has also been used to determine the estrogenicity of sewage effluent and surface water (26-28;131). One drawback which has to be considered when using these types of assays is, however, that antiestrogens which might also be present in the water samples can bind to the estrogen receptor and counteract the estrogenic response. An underestimation of the actual estrogenic potential of the water source might therefore take place. This has been demonstrated earlier (132). Further, several in vitro assays have found approximately the same estrogenicity of 17ß-estradiol and ethinylestradiol while the ethinylestradiol in vivo has been found to have an approximately 10 times higher estrogenicitet than estradiol. This might also lead to an underestimation of the actual feminising potential of a water source. Körner et al. 1999, 2000, 2001 have used the E-screen to assess the estrogenicity of sewage effluent and sludge from German sewage treatment plants (26-28). Analysis of sewage from five different municipal sewage treatment plants in South Germany detected 2-25 ng/l estrogen equivalents (EEQs). The plants all had mechanical purification devices (primary clarification), activated sludge treatment, biological nitrate removal (nitrate/denitrification) and final settlement tanks. In another study EEQs of between 0.2 and 7.8 ng/l EEQ were detected (median 1.6 ng/l) in effluents from 16 municipal and two industrial STPs in the state of Baden-Wüttemberg, Germany (26). This study indicated rather constant inputs of estrogenic substances via STP effluent to the rivers. This is not in agreement with an English study which demonstrated very variable estrogen levels in sewage effluent analysed over a period of 8 months (133). Effluent from a modern municipal STP in Germany with a technical standard reported to be very high still contained 6 ng/l EEQ (28). The municipal STP processed sewage from 200,000 inhabitants and was reported to have a capacity of 350,000 population equivalents (P.E.)11. 60 % of the wastewater was of domestic origin and 40 % of industrial or other origin. Chemical analysis of the effluent water detected 4-tert-octylphenol (4-tOP), 4-nonylphenol (NP) and bisphenol A in the effluent at concentrations from 0.16 to 0.36 m g/l and the contribution to the total estrogenicity of the sewage effluent coming from these compounds was assessed to 0.7-4.3 %. This indicated that most of the estrogenicity of the effluent was due to the presence of natural and synthetic estrogens (28). Since the effluent was diluted to 50 % in the Danube River in vivo estrogenicity on the river fauna was not expected by the authors. Estrogenicity suspected to impact fish has, however, been demonstrated for another large German river, the Rhine, despite a 10-100 times lower YES estrogenicity of the river water compared to the effluent. Chemical analysis detected estradiol at relevant concentrations of 3.9 ng/l in the River Rhine along with some phytosteroids, and estradiol was thought to explain part of the in vitro and in vivo induction of vitellogenin which was obtained by exposure of rainbow trout hepatocytes and male rainbow trout, respectively to river water (134). Natural and synthetic estrogens have also been considered responsible for the observed estrogenicity of sewage effluent in other studies. Sewage effluent from seven STPs which discharged into English Rivers were demonstrated to contain three fractions with estrogenic activity when these were tested with the YES assay (25). Chemical analysis isolated 17b-estradiol, estrone and ethinylestradiol from these fractions. Estradiol and estrone were detected in all samples in concentrations of 1- 48 ng/l and 1 – 76 ng/l, respectively while ethinylestradiol was detected in three effluents at concentrations of 0.2 – 7.0 ng/l. These levels have led to the conclusion that most effects on freshwater feral fish observed in English rivers are a result of natural estrogens excreted from women. The effluent tested in this study contained little or no agricultural input and all natural estrogen input was believed to be of human origin. The feminising effects which have been found in English estuaries in feral flounder also seem to be related to the presence of the natural hormone estradiol in the effluent (129). This was the major component causing 84-90 % of the estrogenicity in a YES assay although the magnitude of the response of a maximum of 24 ng/l EEQ was less than observed in freshwater environments in England. The sediment pore water was also found to have an estrogenic effect equivalent of 7 ng E2/l but the responsible compounds were not identified. The estrogen equivalent levels were also found to be higher in Dutch fresh water compartments and biota compared to coastal and marine environments (116) The estrogenic activity of 2.2 – 12 ng EEQs/l which has been detected by in vitro assays at Meiliang Bay of Taihu Lake - the third largest lake of China – has also been ascribed mainly to estradiol and ethinylestradiol (29). This lake has been described as one of the most polluted water bodies of China and both municipal and industrial sewage from a city of 4 mio inhabitants are discharged into the bay. Estradiol at concentrations from 1.6 – 15.5 ng/l and concentration of ethinylestradiol from 5.7 – 30.8 ng/l have been measured. In effluents from three municipal wastewater treatment plants in Michigan, four point source locations and five locations in Lake Mead, EEQs of 1.90 – 14.9 ng/l, 3.64 – 5.30 ng/l and 1.08 – 10.9 ng/l, respectively have been found by Snyder (30). Estradiol and ethinylestradiol accounted for 88 to 99.5 % of the total estrogen equivalents in the water samples and although alkylphenols were detected in up to 37 m g/l water they were only assessed to contribute to less than 0.5 % of the total EEQ. In a Korean river total estrogenic activity was 0.5 – 7.4 ng EEQ/l in water downstream of sewage discharge with a sediment activity of 3.39 – 10.70 ng EEQ/g (131). In a Japanese river EEQs of 3.5 ng/l have been reported (135). Effluent from two French STPs which received mainly domestic wastewater (80,000 and 300,000 P.E.s, respectively) had a total estrogenic activity of 1.36 - > 8.17 ng EEQ/l, and estrogenic activity of 0.27-1.36 EEQ/l was also detected in receiving river water (136). In a South African study of 25 selected water and sediment samples using the YES assay, estrogenic potencies ranged from below detection limit (0.027 ng/l) to 23.5 ng EEQ/l (137) and from below detection limit (0.020 ng/g) to 13,9 ng/g for the sediment samples. The last results which will be mentioned from studies assessing the in vitro estrogenicity are on sewage and surface water in the Netherlands and Belgium. The Dutch national survey recently reported 0.03-16.01 ng EEQ/l in sewage but only 0.07-0.47 ng EEQ/l in river water. More than 80 % of the estrogenic activity of the effluent could, however, not be explained by chemical analysis of the known (xeno)-estrogens (6;32). A study of the estrogenic activity of Flemish rivers and effluent surprisingly found the highest estrogenic potency in the surface water samples compared to the effluent (33). The estrogenic potency of the water samples ranged from below detection (~ 2.75 ng EEQ/l) to 81 ng EEQ/l. More than 10 ng EEQ/l were found in 7 of 16 samples. As described above the use of in vitro assays has demonstrated estrogenicity of sewage in numerous countries and in a large number of these studies the major part of the in vitro estrogenicity was assigned to the presence of natural and synthetic estrogens in the samples. Therefore the following sections describing environmental concentrations in relation to known dose-response relationships regarding endocrine disrupting effects will concentrate on the natural estrogens 17b -estradiol, estrone and estriol, the synthetic estrogen ethinylestradiol and among xenoestrogens the more potent alkylphenols and bisphenol A.
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