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The proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-Screen assay) was applied for quantitative determination of total estrogenic activity in 24-h composite effluent samples from 16 municipal and two industrial sewage treatment plants (STPs) in the state of Baden-Württemberg, southwestern Germany. The estrogenic efficacy relative to the positive control, 17beta-estradiol, was between 26 and 74% (median, 48%) for the 16 municipal STPs. Estradiol equivalent concentrations (EEQs) were between 0.2 and 7.8 ng/L (median, 1.6 ng/L) and, thereby, were lower than those found in a pilot study, which revealed EEQs of greater than 10 ng/L in the effluents of two other STPs. The EEQs in 14 of the 16 effluent samples were very similar (0.9-3.3 ng/L), indicating a rather constant input of estrogenic substances via STPs into rivers. Additional activated charcoal filtration turned out to be very efficient in further eliminating estrogenic activity from effluents. The EEQs of the E-Screen assay and those calculated from the results of extensive chemical analysis using the estradiol equivalency factors determined for 13 natural and synthetic estrogenic substances were comparable for most of the effluent samples. 17beta-Estradiol, 17alpha-ethinylestradiol, and, to a lesser extent, estrone contributed to 90% or more of the EEQ value.

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The use of recycled paper for the manufacture of food contact materials is widespread, but very little is known about the presence of potential contaminants in the paper. The purpose of this study was to assess the worst-case migration of estrogenic active compounds using extracts of paper for household use. Twenty different brands of kitchen rolls, nine of which were made from recycled paper and the remainder from virgin paper, were obtained from retail shops. Paper extracts were subjected to (a) determination of the total estrogenic activity by using an in vitro estrogen screen based on yeast cells stably transfected with the human estrogen receptor alpha and (b) chemical analysis and quantification by GC/MS, GC/FTIR/MS, and GC/FID for detection of a variety of estrogenic compounds. A marked estrogenic response was observed in nine of the extracts, seven of which were made from recycled paper and two from virgin paper. The chemical analysis revealed that extracts made from recycled paper contained levels of bisphenol A ranging from 0.6 to 24 mg/kg of kitchen roll, whereas extracts from virgin paper contained no bisphenol A or only negligible amounts. In contrast, 4-tert-octylphenol, 4-nonylphenols, and di-n-butyl and diisobutyl phthalate were present to a varying degree in both recycled and virgin paper with no apparent preferable distribution between the two paper types. The estrogenic response of the two extracts made from virgin paper appeared to be due partly to the presence of the preservative propyl paraben. Diisopropylnaphthalene, which turned out to be weakly estrogenic active in vitro (EC(50) = 53 microM), was detected in minor amounts in most of the extracts with the major part, ranging from 0.3 to 4.7 mg/kg of paper, found in recycled paper. Our findings that recycled kitchen rolls contain bisphenol A and other xenoestrogens may apply to other types of recycled paper used for food packaging and emphasize the importance of identifying this and other contaminants in recycled paper in general. These data indicate that bisphenol A may be useful as a purity indicator for recycled paper.