THE ORIGINS AND OCCURRENCE OF ESTROGENIC A-RING AROMATIC STEROIDS IN U.K. SEWAGE TREATMENT WORKS EFFLUENTS
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There is worldwide concern over the possible estrogenic effects of organic chemicals on a variety of wildlife and indeed on humankind. In the U.K., estrogenic compounds in sewage treatment works (STW) effluents have been implicated in causing the increases in egg yolk protein production observed in caged male trout and other fish species. At the initiation of the present study, few of the estrogenic compounds in STW effiuents had been recognised, although circumstantial evidence suggested that steroidal hormones were primary candidates. Cholesterol is abundant in STW effluents and is the precursor of all steroidal hormones biosynthesised in mammalian systems. Thus, the aim of the present study was to examine the hypothesis that cholesterol might undergo A-ring aromatisation, during sewage treatment, producing estrone and 17β-estradiol via, intermediates such as l9-norcholest-l,3,5(10)-trien-3-ol (NCT). To study this hypothesis NCT was first synthesised via a known route and several of its chromatographic and mass spectral properties established for the first time. NCT itself was found to possess some estrogenic potential determined using an established assay but this was rather weak compared to 17β-estradiol - about 200,000 times less active at the minimum concentration needed to invoke a response. NCT also proved to be a much more hydrophobic compound than, for example, 17β-estradiol with a computed log octanol-water partition coefficient (Kow) of over 9 compared with a log Kow of about 4 for l7β-estradiol. The established analytical properties of NCT were then used to investigate possible NCT formation in sewage. Radiolabelled 14C-cholesterol was incubated aerobically and anaerobically in Semi-Continuous Aaivated Sludge (SCAS), Die Away (DA) or simple stand alone STW simulation vessels. The products of incubation in both aqueous and solid fractions were examined by radio-high performance liquid chromatography (r-HPLC), radio-thin layer chromatography (r-TLC) and radio-gas chromatography (r-GC). Aerobic studies showed that side chain cleavage and A-ring rupture of cholesterol occurred rapidly (~25 % of added activity within 24 hrs) as measured by 14C02 evolution. Gaseous evolution was not monitored from the anaerobic experiments. Most remaining activity was associated with the solids fractions in all experiments. In the aqueous experiments both SCAS and DA systems, r-HPLC revealed rapid production of polar products which were not identified further. r-HPLC also revealed non-polar components of which choIest-3,5-diene, an unknown cholestadiene, a cholestadienol (other than 5,7-dienol), cholest-4-en-3-one and possibly NCT were identified by r-GC in the products of DA experiments. Whilst r-HPLC and r-TLC also revealed several products of anaerobic digestion of cholesterol, no compounds were detected by r-GC. STW effluents from two wastewater plants in the North London area were monitored over 7 months for A-ring steroids and other suspected estrogenic chemicals. Both effluents had previously proved estrogenic to caged fish. Liquid and SPM samples were taken, extracted and analysed by GC-MS. The two main estrogens, l7β-estradiol and estrone were identified from all liquid samples but not in SPM extracts. Generally the concentration of estrone (maximum ca 3 ng Lˉ¹) was significantly higher than that for 17β-estradiol (maximum 1 ng fˉ¹). The third natural estrogen, l6a,l7β-estriol, was identified in all Harpenden effluent samples analysed up to a concentration ca 0.5 ng Lˉ¹. However, estriol was not found in Deephams effluent extracts. The phytoestrogen, daidzein, found in soya based products was intermittently found in aqueous effluents samples from both sites >1 ng Lˉ¹. SPM made up <0.001 % of the effluent. Extracts showed that there was a high percentage of steroidal based material with coprostanol>cholesterol=β-sitosterol>>stigmasterol. NCT was twice identified from SPM taken from Deephams with a concentration of 39 and 136 ng 1ˉ¹ but <LOD in Harpenden SPM extracts. In conclusion this study showed that formation of NCT from cholesterol in STW treatment under conditions monitored herein is not a major process. However, estrone, 17β-estradiol and 16a, 17β-estriol are relatively abundant and widespread components of the STW effluents tested. The origins of the components undoubtedly include direct inputs from female urine and faeces but other metabolic sources should not be entirely ruled out.
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