Abstract. This study investigated operational factors influencing the removal of steroid 18 estrogens and alkylphenolic compounds in two sewage treatment works, one a 19 nitrifying/denitrifying activated sludge plant and the other a nitrifying/denitrifying 20 activated sludge plant with phosphorus removal. Removal efficiencies of >90% for 21 steroid estrogens and for longer chain nonylphenol ethoxylates (NP 4-12 EO) were observed 22 at both works, which had equal sludge ages of 13 days. However, the biological activity 23 in terms of milligrams of estrogen removed per tonne of biomass was found to be 50-60% 24 more efficient in the nitrifying/denitrifying activated sludge works compared to the works 25 which additionally incorporated phosphorus removal. A temperature reduction of 6°C 26 had no impact on the removal of free estrogens, but removal of the conjugated estrone-3-27 sulphate was reduced by 20%. The apparent biomass sorption (LogKp) values were 28 greater in the nitrifying/denitrifying works than those in the nitrifying/denitrifying works 29 with phosphorus removal for both steroid estrogens and alkylphenolic compounds 30 possibly indicating a different cell surface structure and therefore microbial population. 31The difference in biological activity (mg tonne -1 ) identified in this study, of up to seven 32 2 times, suggests that there is the potential for enhancing the removal of estrogens and 33 alkylphenols if more detailed knowledge of the factors responsible for these differences 34 can be identified and maximised, thus potentially improving the quality of receiving 35 waters. 36 37 Introduction 38

Due to concerns regarding the release of pharmaceuticals into the environment and the understudied impact of stereochemistry of pharmaceuticals on their fate and biological potency, we focussed in this paper on stereoselective transformation pathways of selected chiral pharmaceuticals (16 pairs) at both microcosm (receiving waters and activated sludge wastewater treatment simulating microcosms) and macrocosm (wastewater treatment plant (WWTP) utilising activated sludge technology and receiving waters) scales in order to test the hypothesis that biodegradation of chiral drugs is stereoselective. Our monitoring programme of a full scale activated sludge WWTP and receiving environment revealed that several chiral drugs, those being marketed mostly as racemates, are present in wastewater and receiving waters enriched with one enantiomeric form (e.g. fluoxetine, mirtazapine, salbutamol, MDMA). This is most likely due to biological metabolic processes occurring in humans and other organisms. Both activated sludge and receiving waters simulating microcosms confirmed our hypothesis that chiral drugs are subject to stereoselective microbial degradation. It led, in this research, to preferential degradation of S-(+)-enantiomers of amphetamines, R-(+)-enantiomers of beta-blockers and S-(+)-enantiomers of antidepressants. In the case of three parent compound - metabolite pairs (venlafaxine - desmethylvenlafaxine, citalopram - desmethylcitalopram and MDMA - MDA), while parent compounds showed higher resistance to both microbial metabolism and photodegradation, their desmethyl metabolites showed much higher degradation rate both in terms of stereoselective metabolic and non-stereoselective photochemical processes. It is also worth noting that metabolites tend to be, as expected, enriched with enantiomers of opposite configuration to their parent compounds, which might have significant toxicological consequences when evaluating the metabolic residues of chiral pollutants.

This paper presents and compares for the first time two chiral LC-QTOF-MS methodologies (utilising CBH and Chirobiotic V columns with cellobiohydrolase and vancomycin as chiral selectors) for the quantification of amphetamine, methamphetamine, MDA (methylenedioxyamphetamine), MDMA (methylenedioxymethamphetamine), propranolol, atenolol, metoprolol, fluoxetine and venlafaxine in river water and sewage effluent. The lowest MDLs (0.3-5.0 ng L(-1) and 1.3-15.1 ng L(-1) for river water and sewage effluent respectively) were observed using the chiral column Chirobiotic V. This is with the exception of methamphetamine and MDMA which had lower MDLs using the CBH column. However, the CBH column resulted in better resolution of enantiomers (R(s)=2.5 for amphetamine compared with R(s)=1.2 with Chirobiotic V). Method recovery rates were typically >80% for both methodologies. Pharmaceuticals and illicit drugs detected and quantified in environmental samples were successfully identified using MS/MS confirmation. In sewage effluent, the total beta-blocker concentrations of propranolol, atenolol and metoprolol were on average 77.0, 1091.0 and 3.6 ng L(-1) thus having EFs (Enantiomeric Fractions) of 0.43, 0.55 and 0.54 respectively. In river water, total propranolol and atenolol was quantified on average at <10.0 ng L(-1). Differences in EF between sewage and river water matrices were evident: venlafaxine was observed with respective EF of 0.43 ± 0.02 and 0.58 ± 0.02.

Here presented for the first time is the enantioselective biodegradation of amphetamine and methamphetamine in river microcosm bioreactors. The aim of this investigation was to test the hypothesis that mechanisms governing the fate of amphetamine and methamphetamine in the environment are mostly stereoselective and biological in nature. Several bioreactors were studied over the duration of 15 days (i) in both biotic and abiotic conditions, (ii) in the dark or exposed to light and (iii) in the presence or absence of suspended particulate matter. Bioreactor samples were analysed using SPE-chiral-LC-(QTOF)MS methodology. This investigation has elucidated the fundamental mechanism for degradation of amphetamine and methamphetamine as being predominantly biological in origin. Furthermore, stereoselectivity and changes in enantiomeric fraction (EF) were only observed under biotic conditions. Neither amphetamine nor methamphetamine appeared to demonstrate adsorption to suspended particulate matter. Our experiments also demonstrated that amphetamine and methamphetamine were photo-stable. Illicit drugs are present in the environment at low concentrations but due to their pseudo-persistence and non-racemic behaviour, with two enantiomers revealing significantly different potency (and potentially different toxicity towards aquatic organisms) the risk posed by illicit drugs in the environment should not be under- or over-estimated. The above results demonstrate the need for re-evaluation of the procedures utilised in environmental risk assessment, which currently do not recognise the importance of the phenomenon of chirality in pharmacologically active compounds.

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This paper aims to understand enantioselective transformation of amphetamine, methamphetamine, MDMA (3,4-methylenedioxy-methamphetamine) and MDA (3,4-methylenedioxyamphetamine) during wastewater treatment and in receiving waters. In order to undertake a comprehensive evaluation of the processes occurring, stereoselective transformation of amphetamine-like compounds was studied, for the first time, in controlled laboratory experiments: receiving water and activated sludge simulating microcosm systems. The results demonstrated that stereoselective degradation, via microbial metabolic processes favouring S-(+)-enantiomer, occurred in all studied amphetamine-based compounds in activated sludge simulating microcosms. R-(-)-enantiomers were not degraded (or their degradation was limited) which proves their more recalcitrant nature. Out of all four amphetamine-like compounds studied, amphetamine was the most susceptible to biodegradation. It was followed by MDMA and methamphetamine. Photochemical processes facilitated degradation of MDMA and methamphetamine but they were not, as expected, stereoselective. Preferential biodegradation of S-(+)-methamphetamine led to the formation of S-(+)-amphetamine. Racemic MDMA was stereoselectively biodegraded by activated sludge which led to its enrichment with R-(-)-enantiomer and formation of S-(+)-MDA. Interestingly, there was only mild stereoselectivity observed during MDMA degradation in rivers. This might be due to different microbial communities utilised during activated sludge treatment and those present in the environment. Kinetic studies confirmed the recalcitrant nature of MDMA.

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The impact of loading and organic composition on the fate of alkylphenolic compounds in the activated sludge plant (ASP) has been studied. Three ASP designs comprising carbonaceous, carbonaceous/nitrification, and carbonaceous/nitrification/denitrification treatment were examined to demonstrate the impact of increasing levels of process complexity and to incorporate a spectrum of loading conditions. Based on mass balance, overall biodegradation efficiencies for nonylphenol ethoxylates (NPEOs), short chain carboxylates (NP(1-3)EC) and nonylphenol (NP) were 37%, 59%, and 27% for the carbonaceous, carbonaceous/nitrification, and carbonaceous/nitrification/denitrification ASP, respectively. The presence of a rich community of ammonia oxidizing bacteria does not necessarily facilitate effective alkylphenolic compound degradation. However, a clear correlation between alkylphenolic compound loading and long chain ethoxylate compound biodegradation was determined at the three ASPs, indicating that at higher initial alkylphenolic compound concentrations (or load), greater ethoxylate biotransformation can occur. In addition, the impact of settled sewage organic composition on alkylphenolic compound removal was evaluated. A correlation between the ratio of chemical oxygen demand (COD) to alkylphenolic compound concentration and biomass activity was determined, demonstrating the inhibiting effect of bulk organic matter on alkylphenol polyethoxylate transformation activity. At all three ASPs the biodegradation pathway proposed involves the preferential biodegradation of the amphiphilic ethoxylated compounds, after which the preferential attack of the lipophilic akylphenol moiety occurs. The extent of ethoxylate biodegradation is driven by the initial alkylphenolic compound concentration and the proportion of COD constituted by the alkylphenol polyethoxylates (APEOs) and their metabolites relative to the bulk organic concentration of the sewage composed of proteins, acids, fats, and polysaccharides. Secondary effluents from this study are characterized by low bulk organic concentrations and comparatively high micropollutant concentrations. Based on the biodegradation mechanism proposed in this study, application of high rate tertiary biological treatment processes to secondary effluents characterized by low bulk organic concentrations and comparatively high APEO concentrations is predicted to provide a sustainable solution to micropollutant removal.