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1 2 3 4 The white-rot fungus Trametes versicolor reduces the estrogenic activity of a mixture of emerging 5 contaminants in wastewater treatment plant effluent 6 7 Abstract 57 58This study investigated the removal of common emerging contaminants (ECs) and related estrogenic activity from 59 wastewater effluent by a strain of the white-rot fungus Trametes versicolor with previously unreported 60 bioremediation potential. T. versicolor NRRL 66313 was grown in carbon-amended sterile secondary wastewater 61 treatment plant (WWTP) effluent. Aerated batch reactors containing sterile, glucose-amended (5 g l -1 ) wastewater 62 were inoculated, incubated for eight days, and then spiked with either 5 mg l -1 of 17β-estradiol (E2), or a mixture of 63 E2, atrazine (ATZ), bisphenol A (BPA), carbamazepine (CBZ), N,N-diethyl-3-methylbenzamide (DEET), estrone 64 (E1), 17α-ethynylestradiol (EE2), oxybenzone (OBZ), and triclosan (TCS) to a final concentration of 350 µg l -1 65 each. Abiotic and heat-killed fungus controls were also prepared. EC and metabolite concentrations were measured 66 over time using HPLC and Gas Chromatography coupled with Time-Of-Flight Mass Spectrometry (GC-TOFMS). 67Estrogenic activity was measured on the same samples using the Yeast Estrogen Screen. In less than 5 hours, active 68 fungi decreased the concentration of E2 from 5 mg l -1 to below detection, producing E1 as a metabolite and 69 subsequently removing it from solution. Acting on the mixture of ECs, fungi removed BPA, E1, E2, and EE2 to a 70 significant degree (62-100%) vs. controls (0-28%) in only 3.5 hours, reducing the estrogenic activity of the mixture 71 by 77% (vs. 4-8% for controls). After 12 hours, the total reduction of estrogenic activity was greater than 98% (vs. 72 24-42% for controls). These results show that T. versicolor NRRL 66313 can reduce the estrogenic activity of 73 mixtures of estrogens and BPA, which are typically the most significant contributors to the hormone disrupting 74 activity of domestic WWTP effluent. 75 76 77

An innovative form of Fisher ratio (F-ratio) analysis (FRA) is developed for use with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC−TOFMS) data and applied to the investigation of the changes in the metabolome in human plasma for patients with injury to their anterior cruciate ligament (ACL). Specifically, FRA provides a supervised discovery of metabolites that express a statistically significant variance in a two-sample class comparison: patients and healthy controls. The standard F-ratio utilizes the between-class variance relative to the pooled within-class variance. Because standard FRA is adversely impacted by metabolites expressed with a large within-class variance in the patient class, "control-normalized FRA" has been developed to provide complementary information, by normalizing the between-class variance to the variance of the control class only. Thirty plasma samples from patients who recently suffered from an ACL injury, along with matched controls, were subjected to GC × GC−TOFMS analysis. Following both standard and control-normalized FRA, the concentration ratio for the top 30 "hits" in each comparison was obtained and then t-tested for statistical significance. Twenty four out of 30 metabolites plus the therapeutic agent, naproxen (24/30), passed the t-test for the control-normalized FRA, which included 8/24 unique to control-normalized FRA and 16/24 in common with the standard FRA. Likewise, standard FRA provided 21/30 metabolites passing the t-test, with 5/21 undiscovered by control-normalized FRA. The complementary information obtained by both F-ratio analyses demonstrates the general utility of the new approach for a variety of applications.