WP001 (NOT-1117-838620) Examination of Estrogenic Compounds in Wastewater Treatment Influent and Effluent.
Start time: 8:00 AM
Notch, E¹, Mayer, G¹, ¹ University of Maine
Recently, much attention has been given to endocrine active compounds in the environment. One class of xenoestrogens includes the synthetic estrogens commonly found in pharmaceuticals such as oral contraceptives and hormone replacement therapies. Synthetic estrogens such as these mimic natural estrogens well at the receptor level, but are more resistant to degradation by natural processes. Because of its greater stability and higher potency in vivo, EE2 may be of disproportional toxicological importance despite being found at much lower concentrations than natural steroids such as E2 and E1. It has been shown that conjugated forms of estrogens can be activated during wastewater treatment processes. We screen influent and effluent from multiple wastewater treatment plants that utilize different treatment technologies with an in vitro biological assay. This bioassay utilizes the activation of the Xenopus vitellogenin promoter and the subsequent expression of luciferase as a surrogate for whole animal estrogen response.

WP002 (RIO-1117-840187) Rainbow trout as an indicator species of endocrine disruption in California surface waters.
Start time: 8:00 AM
Riordan, D.¹, Biales, A.², de Vlaming, V.¹, Markiewicz, D.¹, Holmes, R.³, Otis, P.⁴, ¹ University of California, Davis, Aquatic Toxicology Laboratory, Davis, CA² US EPA, National Exposure Research Laboratory, Molecular Indicator Research Branch, Cincinnati, OH³ Regional Water Quality Control Board, Region 5, Sacramento, CA⁴ Regional Water Quality Control Board, Region 1, Santa Rosa, CA
There is substantial evidence documenting the existence of endocrine disrupting chemicals (EDCs) in surface waters. Our goal is to develop and apply a short-exposure and economical method capable of detecting low concentrations of estrogenic EDCs in these waters. Our procedure involves exposing larval rainbow trout (Oncorhynchus mykiss) to water samples and analyzing their livers for vitellogenin mRNA (Vg) using SYBR Green or TaqMan® RT-qPCR (reverse-transcription quantitative polymerase chain reaction). Procedure development experiments with the synthetic estrogen 17-ethinyl estradiol (EE2) indicated that concentrations as low as 10 ng/L of EE2 would up-regulate the Vg gene. Within treatment variability, however, was high. In an effort to reduce this variability, a series of experiments conducted in which test temperature, test chamber size, number of replicates per treatment and number of fish per replicate were altered. The most significant effect arose from increasing the number of fish per replicate. Liver samples from each of the three fish in a test chamber were pooled and analyzed by PCR as one sample. The result was the acquisition of data that were more normally distributed, and in some cases, allowed for parametric, rather than non-parametric statistical analyses. Currently, we are conducting a series of experiments with ambient surface water samples from various regions of California. Early findings indicate the presence of estrogenic EDCs. Follow-up studies with ambient samples, in conjunction with additional procedure development, are scheduled for summer, 2005.

WP003 (HID-1118-032633) Induction of Estrogenic Activitiy in the Non-Cropping Paddy Field Receiving a Rural Sewage Effluent.
Start time: 8:00 AM
Hida, Y¹, Kurata, T¹, Yamada, Y, Kunimatsu, T¹, Tatarazako, N⁴, Osaki, S², Fukui, H³, Shiraishi, F⁴, Shiraishi, H⁴, ¹ Department of Ecosystem Studies, School of Environmental Science, The University of Shiga Prefecture, Hikone, Japan⁴ National Institute for Environmental Studies, Tsukuba, Japan² Shiga Prefectural Federation of Land Improvement Association, Otsu, Japan³ Shiga Prefectural Government, Otsu, Japan
Last year, we reported the estrogenic activities of a rural sewage effluent, which were rarely detected, appeared after the effluent flowed into the non-cropping paddy field. In this study, we analyzed the mechanism of increase in estrogenic activity and investigated estrogenic action to caged medaka (Oryzias latipes) in the non-cropping paddy field environment. The whole effluent of 84 m³/d from a rural sewage was inflowed everyday into the non-cropping paddy field (830 m²) involved the division of the canal into seven areas. Watercress and round crucian carp (funa) are introduced into the fourth and fifth divisions, respectively. We collected surface waters at an effluent entry place and seven various distances downstream, the end place of each division, in the canal every two weeks from April 2003 to September 2004. We evaluated estrogenic activity of each sample using assay with the modified yeast two-hybrid assay system (human and medaka estrogen receptors: hER and medER). The hER assay system is high sensitive specifically to natural estrogens, and the medER assay system, by contrast, has low sensitivity to natural estrogens, and is relatively high sensitive to estrogenic industrial chemicals and phytoestrogens, the so-called xenoestrogens. To assess the estrogenicity of non-cropping paddy field environment receiving effluent, the monitoring system for plasma vitellogenin production and induction of hepatic cytochrome P4501A and 2B activities, measured as the O-dealkylation of ethoxy-, pentoxy- resorufin, in caged medaka was used. Estrogenic activities on the medER assay system of surface water at the end place of third division in the canal increased during spring and summer and decreased during autumn and winter. This temporal variation was contrary to that of dissolved oxygen solubility, and that indicates there exists some relationship between estrogenic activity and dissolved oxygen solubility. No estrogenic effect on caged medaka, however, was observed.

WP004 (KOS-1117-751363) An Informatic Approach to Estimating Ecological Risks Posed by Pharmaceutical Use.
Start time: 8:00 AM
Kostich, M¹, Lazorchak, J¹, Toth, G¹, ¹ US Environmental Protection Agency, Cincinnati, OH, USA
Pharmaceuticals administered to humans and other animals are often excreted from treated organisms as intact drug or as active metabolites. Some of these active materials have been shown to make their way into the environment. However, the environmental concentrations of the vast majority of pharmaceuticals and their metabolites are not known. The sensitivity of native organisms to chronic exposure to the doses of active material likely to be found in the environment is also not known. Direct determination of these important parameters is too expensive to perform on the entire pharmacopoeia, and a rational way of prioritizing individual drugs for more detailed study is needed. For many drugs sufficient information is available in the scientific literature and within regulatory filings to estimate the upper bound of environmental concentrations and most probable mechanisms of environmental toxicity. Although these estimates involve large confidence intervals, they are quickly and cheaply produced, and are probably the best available criteria for prioritizing drugs for more expensive direct tests of environmental impact. Here we describe a new method similar to that employed by the EU and by the US FDA for estimating risks of human prescription pharmaceuticals based on information found in regulatory filings as well as scientific and trade literature. Available data on usage patterns, metabolic transformation, and physical/chemical properties are fed into models of dilution, degradation, partition between matrices, and bioavailability in order to estimate effective environmental concentrations. Available data on mechanisms of action and modes of toxicity are considered together with cursory phylogenetic analysis to estimate the sensitivity of select native organisms to the estimated highest likely environmental concentrations of each drug. Scores produced by this procedure will be used to prioritize pharmaceuticals for more detailed analytical and toxicological follow-up. The applicability of modified versions of this method to over-the-counter pharmaceuticals and veterinary pharmaceuticals is also described. Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

WP006 (STA-1117-828957) A weight of evidence assessment of the fate of alkylphenol ethoxylates and their biodegradation intermediates in the environment.
Start time: 8:00 AM
Staples, C¹, Klecka, G², Naylor, C³, ¹ Assessment Technologies, Inc, Spotsylvania, Virginia, USA² The Dow Chemical Company, Midland, Michigan, USA³ CGN, Austin, Texas, USA
Alkylphenol ethoxylates (APEs) are widely used surfactants in the US in such applications as industrial and institutional cleaners, production of pulp and paper, textiles, and as emulsifying agents, dispersants and adjuvants. The most common APEs are nonylphenol ethoxylates (NPE) and octylphenol ethoxylates. Most uses involve the release of spent surfactant into wastewater treatment systems. After biological degradation and removal, trace levels of the parent surfactants and their biodegradation intermediates can be detected in effluents from wastewater treatment plants and in downstream surface waters and sediments. In addition, NPEs adsorbed to de-watered sludge may enter the terrestrial environment when the sludge is used as a nutrient supplement (i.e., biosolids) applied to soil. Studies have been conducted that measured the primary and ultimate biodegradation of NPEs and OPEs in order to assess their potential fate in the environment. Commercial products such as NPE-4 and NPE-9 are actually mixtures of numerous oligomers ranging from NPE-1 to NPE-20 or higher, but averaging 4 and 9 ethoxylate units, respectively. Data are available for the commercial products themselves, as well as individual lower ethoxylates, ether carboxylates (APECs), and the alkylphenols themselves. The database includes tests conducted using standard OECD protocols, freshwater and marine die-away methods (with and without sediment, die-away methods in soil, and field studies. The database is presented here and used in a weight of evidence assessment of the degradability of these substances. The weight of evidence shows that commercial APEs, their degradation intermediates (lower APEs and APECs), and APs are both primarily and ultimately degraded in the environment and do not meet internationally determined criteria for being designated persistent substances.

WP007 (STA-1117-829901) The Relative Toxicity of Nonylphenol Ethoxylates and their Biodegradation Intermediates.
Start time: 8:00 AM
Staples, C¹, Losey, B², ¹ Assessment Technologies, Inc., Spotsylvania, Virginia, USA² RegNet Environmental Services, Inc., Washington, DC, USA
An extensive aquatic toxicity database exists for nonylphenol ethoxylates (NPE) and their degradation intermediates: low mole NPEs, nonylphenol ether carboxylates (NPEC) and nonylphenol (NP). Regulators have used the database to define the relative toxicity among these compounds in order to assess potential cumulative risks to aquatic organisms exposed to a mixture of the compounds while acknowledging conflicting results and data gaps in the low mole range of NPEs (i.e. NPE2-NPE8). Relative toxicity has been assessed using two types of endpoints: a) conventional endpoints of survival, growth and development, and reproduction, and b) potential indicators or biomarkers of endocrine modulation (e.g., vitellogenin induction). Recent work with rainbow trout (Oncorhynchus mykiss) has clarified prior discrepancies concerning the potency of the various compounds for vitellogenin induction. These findings suggest that the potencies of the various substances were consistent with relative toxicities of these compounds as determined by conventional endpoints. Further, in the absence of available data on the relative toxicity of the lower mole NPE2-8 compounds, it had been assumed that all lower NPE<9 were approximately as toxic as NPE1. The relative toxicity of NP, lower mole NPEs, higher mole NPEs and NPECs has been recently clarified by studies with medaka fish (Oryzias latipes) which monitored effects on growth, development and sex ratio endpoints. The data show that the lower mole NPE have similar toxicity to higher mole NPEs, rather than the more toxic NPE1. This presentation will explain how these new data provide the basis for refining the existing assessment of the relative toxicities between NP, NPE1, NPE2-8, NPE9, and NPECs. In addition, the refined relative toxicities of NPE biodegradation intermediates are used in a companion presentation that evaluates the potential exposure and environmental risks of these substances in US waters.

WP008 (WIL-1117-835086) Assessment of toxicological potential in marine coastal areas using YES assay and chemical analyses.
Start time: 8:00 AM
Willett, K¹, Chaudhary, A¹, Khan, S¹, Singh, S², Azua, A², Gardinali, P², ¹ University of Mississippi, University, MS, USA² Florida International University, Miami, FL, USA
The goal of this research is to assess the potential toxicological impacts of degraded water quality due to human derived activities in marine coastal areas. Specifically, samples were collected along the Black Creek Canal (C-1) located in South Miami-Dade County which runs through agricultural and urban areas before intersecting Biscayne Bay at Black Point Marina. The canal runs past one of the largest landfills in South Florida and a municipal wastewater plant serving a large urban setting. In this study, fourteen samples from different locations along the C-1 canal were tested chemically for 5 pharmaceuticals and 14 steroids and in the in vitro yeast estrogen screen (YES). YES is used to determine estrogen binding ability (estrogen equivalence) of compounds that are potential endocrine disruptors. At the C-1 canal, estrogen equivalents of the samples were non-detected (ND) - 223 pg/L, and estrone (0.37 - 0.85 ng/L), caffeine (ND - 23 ng/L) and DEET (3.8 - 27 ng/L) were detected. Coprostanol (3.6 - 5.8 ng/L) and triclosan (2.6 - 4.6 ng/L) were detected, however no nonylphenol, bisphenol A, or ethynylestradiol were detected. Standards were also tested by YES assay to determine their estrogen equivalence. Estrone and equilin, showed activity in YES assay, with estrogen equivalencies of 0.4 and 0.1 respectively whereas caffeine, DEET and cholesterol showed no activity. The estrogen equivalents detected at C-1 canal were significantly lower than what we have found at other wastewater impacted marine sites (up to 1.3 ng/L). The levels of human-derived contaminants found at C-1 canal do not indicate high potential risk to the ecosystem. However, their presence should not be downplayed because steroid hormones have the potential to cause endocrine disruption in sensitive native marine organisms at low ppt concentrations. (Supported by NOAA/NIUST).

WP009 (SIN-1117-835459) A comprehensive method for the fast screening of selected EPOCs in ground, surface and waste waters.
Start time: 8:00 AM
Singh, Simrat¹, Gardinali, Piero^{1, 2}, ¹ Florida International University, Miami, Florida, USA² Southeast Environmental Research Center (SERC), Miami, Florida, USA
The release of steroids and pharmaceuticals into the aquatic environment has been extensively documented and studied in recent years. Because of the increased need for reclaimed water it is important to provide a comprehensive and fast screening tool to monitor EPOCs effectively in a large number of samples. Rather than trying to analyze a wide variety of target analytes at higher concentrations this work presents a simple, fast and comprehensive method for the analysis of 20 of the most relevant wastewater contaminants (EPOCs) in a single extraction. The method uses five surrogate standards (-estradiol-d5, progestrone-d9, estrone-d4, caffeine-12c3, and bisphenolA-d16) for the quantification of five of the most commonly detected pharmaceuticals and 15 hormones and steroids. The method involves liquid-liquid extraction of the water sample with methylene chloride, concentration of the final extracts using a stream of clean nitrogen and derivatization using BSTFA. Finally, 2l of the final extract are injected into a Finnigan Trace DSQ GC-MS instrument equipped with a DB-5MS column (30m x 0.25mm x 0.25 m film thickness) and analyzed using a single quadruple mass detector operated in SIM mode. The calculated Method Detection Limit (MDL) for all the compounds analyzed is in between 0.1-2.0 ng/L. The method was applied to treated wastewater and surface water samples collected from different locations in Florida. In general, the results showed that water collected from inland canals and marinas had low concentrations of multiple steroids, pharmaceuticals, and personal care products. The compounds more frequently detected in surface waters were: coprostanol, cholesterol, estrone, caffeine, triclosan and DEET. The concentration ranges of estrone and -estradiol in surface waters were from 0.31 to 5.25 ng/L and the concentration ranges of caffeine and triclosan were between 1.74 to 67.7 ng/L. The concentration ranges for the analytes detected in the treated wastewater effluents were higher (i.e. for estrone it was 1.96 to 65.1 ng/L and for -estradiol it was 0.3 to 6.4 ng/L) and dependent on the treatment processes used to achieve the final effluent.

WP010 (GOD-1117-794864) Development of ELISAs for the analysis of EDCs in Wastewater Treatment Effluents.
Start time: 8:00 AM
Goda, Y¹, Kitaoka, K², Hirobe, M¹, Fujimoto, S¹, Okayasu, Y³, Ohkawa, H⁴, Shiraishi, H⁵, Rubio, F⁶, Ike, M⁷, Fujita, M⁷, ¹ Japan EnviroChemicals Ltd., Osaka, Japan² Japan EnviroChemicals Ltd., Tokyo, Japan³ Independent Admimistrative Institution Public Works Research Institute, Tsukuba, Japan⁴ Fukuyama University, Fukuyama, Japan⁵ National Institute for Environmental Studies, Tsukuba, Japan⁶ Abraxis LLC., PA, USA⁷ Graduate School of Engineering, Osaka University, Suita, Japan
For the analysis of endocrine disrupting chemicals (EDCs) and estrogens in wastewater treatment effluents, highly sensitive analytical methods are required, because these contaminants are present at very low concentrations, namely ng/L level. Instrumental analysis such as high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) are generally employed for these analysis, however, they have several potential drawbacks such that they need expensive instrumentation, extensive purification and expertise in operation. Therefore, there is a strong need for rapid, simple and cost-effective methods for quantitative analysis of these contaminants. In this study, we have developed 10 kinds of enzyme-linked immunosorbent assay (ELISA) for estrogens [17beta-estradiol (E2), estrone (E1), total estrogen (ES: E1+E2+estriol (E3)), 17alfa-ethynylestradiol (EE2)], endocrine disrupting chemicals [alkylphenol (AP), AP+alkylphenol ethoxylate (AP+APE), and bisphenol A (BPA)] and surfactants [linear alkylbenzene sulfonates (LAS), alkyl ethoxylates (AE), and (APE)]. To validate accuracy of these ELISAs, the comparative studies were conducted between ELISAs (in terms E2 and EE2 analysis, commercially available ELISA kits were also used) and instrumental analysis with the environmental water samples including wastewater treatment effluents. Good correlations were observed in all cases, and regarding E2 and EE2 analysis, the values obtained with the developed ELISAs were most approximated to those with LC-MS/MS than any other commercially available kits. It was also confirmed that combining Nexus solid phase extraction column and dichloromethane elution gave an almost complete recovery of BPA, AP, E1, E2 and EE2 in environmental water samples.

WP011 (SHA-1117-815088) Urban Discharges of Pharmaceuticals and Personal Products to the Tidal Anacostia River (Washington, DC).
Start time: 8:00 AM
Shala, L¹, Foster, G², ¹ George mason University, Fairfax, VA, USA²
Pharmaceuticals are basically commercial drugs and medicines that are taken to treat illness, disease, and medical conditions in both humans and animals. They include antidepressants, psychiatric drugs, chemotherapy drugs, anti-inflammatories, tranquilizers, antihypertensives, antiseptics, lipid regulators, oral contraceptives, synthetic hormones, antibiotics, drugs for enhancing sexual performance and many other classes and types of drugs. In the other heand personal care products include a wide variety of compounds, such as perfumes, shampoos, hair dyes, hair sprays, deodorants, musks, oral hygiene products, make up, nail polish, sun screens, body lotions, lipsticks, and cremes. Recently the fate of pharmaceuticals and personal care products (PPCPs) has received increasing attention as contaminants worldwide. A modified developed analytical method was used to investigate the detection, occurrence and transport of PPCPs (ibuprofen, caffeine, naproxen, triclosan, ketoprofen, bisphenol A, carbamazepine and diclofenac) in storm water samples in the Anacosita River, Washington, DC area. The river water samples are analyzed for PPCPs in water and filtered solids (suspended particles) at low parts per billion (ppb) concentrations. The compounds were extracted from water samples using Oasis HLB solid-phase extraction columns (SPE), derivatized with N,O-bis [Trimethylsilyl] trifluoroacetamide (BSTFA) and analyzed using gas chromatography-mass spectrometry (GC-MS). Suspended particles were previously sonicated and further analyzed in the same way as liquid portion.

WP013 (AND-1117-821873) New Tools for Evaluating Potential Risk of Pharmaceuticals in the Environment: A Case Study.
Start time: 8:00 AM
Anderson, P¹, Buzby, M², Caldwell, D⁴, DuPlessie, B¹, Johnston, J³, ¹ AMEC Earth & Environmental, Westford, MA, US² Merck & Co., Inc⁴ Johnson and Johnson³ Wyeth
Relatively recently researchers have detected low levels of active pharmaceutical ingredients (APIs) in US surface waters. Concomitant with this discovery, not surprisingly, has been a dramatic increase in the number of peer-reviewed articles reporting on the aquatic effects of APIs. While the publication of surface water concentration data and of effects data lays the foundation for evaluating the potential for adverse effects in US surface waters, actually completing such evaluations can be quite resource intensive because the information can be disparate. To facilitate such evaluations, the Pharmaceutical Research and Manufacturers of America (PhRMA) has developed two tools. The first is a model (PhATE^TM) to estimate concentrations of APIs in surface waters that result from patient use of medicines. PhATE^TM has the advantage over relying on measured concentrations of being able to predict surface water concentrations in many more reaches of rivers and under different flow regimes than can be practically measured. The second tool is an aquatic effects database. This database is updated quarterly and will eventually summarize all of the peer-reviewed aquatic effects literature (as of March 2005 more than 750 articles) available on APIs. Finally, it is possible to combine the detected and predicted surface water concentrations with the aquatic toxicity information to evaluate the potential risk. This paper presents an overview of these two tools and presents a case study.

WP014 (COL-1116-988726) A Rapid Method to Quantify Estrogenic Compounds in Wastewater with Recombinant Yeast.
Start time: 8:00 AM
Colosi, J,
Many studies use recombinant yeast Saccharomyces cerevisiae with the human estrogen receptor and a reporter gene to quantify estrogenic compounds in wastewater. The usual protocol is to filter 3 or 4 liters of water through a series of filters followed by solid phase extraction. The estrogenic compounds are eluted from the solid phase extraction disk, dried, and dissolved in DMSO. The yeast is incubated in the presence of a small quantity of the concentrate, ie 1.0 microliter per ml of medium, and the response of the reporter system of the yeast is tested after incubation. We have modified the protocol so that only 50 ml of wastewater is filter sterilized with a bacteriological filter and then the yeast is grown directly in the wastewater that is diluted by half with 2X medium. The processing time of the wastewater is reduced from over 4 hours to less than 15 minutes. We have measured estradiol-equivalent concentrations down to 10 parts per trillion in wastewater by this method. The range of estradiol equivalents in influent of wastewater treatment plants near Easton, Pennsylvania ranged from less than 10 to 54 parts per trillion and in effluent from less then 10 to 20 parts per trillion.

WP015 (SEG-1117-155228) Evaluation of the fate of antimicrobials in the Montréal region.
Start time: 8:00 AM
Segura, P.¹, Lajeunesse, A.², Gagnon, C.², Sauvé, S.¹, ¹ Université de Montréal, Montréal, QC, Canada² Environnement Canada-Centre Saint-Laurent, Montréal, QC, Canada
A method using solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry detection (LC/MS/MS) was developed to study the fate of the most prescribed antimicrobials in the Montréal region (Québec, Canada). We analyzed samples of influents and effluents from the sewage treatment plant (STP) as well as surface water sampled in the outfall and its nearby zones on the St. Lawrence River. Our preliminary findings indicate the presence of the fluoroquinolone antimicrobials in the STP effluent with ciprofloxacin being the most abundant (41 ± 18 ng/L) among those antimicrobials. The efficiency of the STP to eliminate the targeted compounds was evaluated from our results. Their distribution and environmental fate were also determined in the effluent receiving waters.

WP016 (BAT-1117-730202) Influence of Municipal Treatment Plant Design on the Removal Efficiency of Antibiotics from Wastewaters.
Start time: 8:00 AM
Batt, Angela¹, Kim, Sungpyo¹, Aga, Diana¹, ¹ State University of New York at Buffalo, Buffalo, New York, United States
Samples from various stages of treatment from five full-scale wastewater treatment plants (WWTPs) employing different technologies and designs were analyzed for antibiotics and caffeine. The WWTPs chosen utilized a variety of secondary removal processes, such as a two stage activated sludge process with nitrification, extended aeration, rotating biological contactors, pure oxygen activated sludge, and carbon adsorption. Advanced treatment processes, such as sand filtration, metal salt precipitation, and chemical disinfection were also incorporated in some of the WWTPs. Among the antibiotics detected were sulfamethoxazole, trimethoprim, ciprofloxacin, and tetracycline at concentrations ranging from to 0.061 to 7.9 g/L. Caffeine was detected in the influent or primary effluent of all WWTPs at concentrations above the dynamic range of the method (1000 g/L) and in the final effluent at concentrations ranging from 0.56 to 132 g/L. The antibiotics removal efficiencies by each treatment process, along with the overall removal efficiencies of each municipal WWTP were determined. The results from this study give insight on how the WWTP design and operation parameters, such as solids retention time (SRT) and hydraulic retention time (HRT), affect the removal of antibiotics from wastewater.

WP017 (BEN-1117-734863) Acidic pharmaceuticals in surface waters of selected Ontario watersheds.
Start time: 8:00 AM
Bennie, D¹, Struger, J¹, ¹ Environment Canada, Burlington, ON, Canada
Pharmaceutical products are used to control human diseases and conditions as well as enhance animal health and increase food production efficiency. These substances enter the environment via STP effluent discharges, land application of sewage sludges and agricultural animal wastes. This study set out to determine the temporal occurrence of acidic pharmaceuticals in surface waters at 6 sites in Hamilton Harbour (HH) and 6 sites in the Grand River in southern Ontario. Both watersheds receive multiple inputs from STPs. Monthly samples were collected from these locations from May 2003 to May 2004 and were analyzed for 12 acidic pharmaceuticals. Preliminary data shows four pharmaceuticals were detected regularly at levels above analytical detection limits. Those substances were ibuprofen, gemfibrozil, naproxen and triclosan. The maximum concentrations found in the study were 1590, 103, 753 and 428 ng/L, respectively. Other detectable substances include clofibric acid, salicylic acid, diclofenac, indomethacin and fenofibrate. In HH, concentrations of pharmaceuticals decrease significantly in the gradient from the STPs to the Harbour to Lake Ontario. Distribution patterns on the Grand River are different due to the increased number of STP discharge contributions at periodic intervals to the River.

WP018 (SAX-1117-764286) Critical Analysis of the Proposed European Framework for Ecological Assessment of Pharmaceuticals.
Start time: 8:00 AM
Saxe, Jennifer¹, Sharma, Manu¹, Ward, Timothy¹, ¹ Gradient Corporation, Cambridge, MA, USA
In January 2005, the European Medicines Agency issued a revised draft "Guideline on Environmental Risk Assessment of Medicinal Products for Human Use." This guideline prescribes a series of risk assessment steps that pharmaceutical manufacturers' scientific experts must undertake as part of the drug registration process to evaluate the potential for active pharmaceutical ingredients (APIs) to result in ecological effects when discharged in wastewater or via land application of API-containing biosolids. Although the final guideline is still being developed, there is a need for API manufacturers to closely monitor the proposed guideline for several reasons. (1) Many of the tests differ from those typically included in environmental fate and effects test batteries currently used for APIs; for example, chronic toxicity testing is proposed in lieu of acute testing. Thus, results of API testing currently done would not be valid for an assessment under the proposed guideline. (2) Many of the tests in the guideline require months to complete, so planning product registration timelines in the future would be impacted. (3) The aggregate cost of the tests required for many APIs would be in excess of U.S. $200,000, so future product registration budgets would be impacted. Because of these important consequences of the guideline, a critical evaluation of the testing and assessment protocol is provided. Specifically, testing and assessment steps that might be streamlined or avoided by efficiently leveraging all available data are described. For example, OECD Test 308, a sediment transformation test, lasts up to 100 days at cost of about U.S. $70,000, and is required by the guideline for almost all new APIs. The results of this test are used only to decide whether to undertake further sediment and persistent/bioaccumulative chemical assessment. This same decision could be effectively made using the results of less costly and time-consuming tests.

WP019 (GRA-1117-833014) Fate of environmental estrogens in a wastewater-dominated stream with feminized fish populations.
Start time: 8:00 AM
Gray, James¹, Vajda, Alan ², Woodling, John², Norris, David ², Barber, Larry ¹, ¹ US Geological Survey, Boulder, CO, USA² University of Colorado, Boulder, Boulder, CO
Wastewater impacted streams contain biogenic contaminants, such as 17-beta-estradiol (E2), that can elicit endocrine disrupting effects in fish at low concentrations (1 ng/L), and industrial chemicals, such as nonylphenol (NP), that induce similar effects at higher concentrations (>1000 ng/L). Furthermore, a variety of compounds found in wastewater effluents including steroids, detergents and certain human pharmaceuticals are known to be biologically active but have unknown effects at environmentally relevant concentrations. A Lagrangian sampling protocol was employed in conjunction with analysis of intrinsic geochemical tracers and a conservative tracer pulse to characterize contaminant hydrology in Boulder Creek, CO downstream of a wastewater treatment plant (WWTP) discharge. Ongoing work presented in a related abstract shows that white suckers (Catostomus commersoni) congregating downstream of the WWTP were feminized relative to upstream populations. Induction of vitellogenin and intersex gonadal tissue was only observed downstream of the WWTP and the sex ratio (85% female) was no longer evenly distributed. Although E2 and NP concentration gradients between the upstream and downstream sites were steep, other environmental factors also may contribute to the apparent feminization of white suckers below the WWTP. To account for these factors, juvenile white suckers and fathead minnows (Pimephales promelas) will be exposed to wastewater effluent and upstream water in flow-through aquaria where environmental variables will be controlled. These aquaria will be contained in an on-site laboratory, so fish will experience actual environmental exposures over the course of a 28-day experiment. During the course of the experiment, levels of endocrine disrupting chemicals in the aquaria will be monitored in order to establish a causative link between chemical exposure and abnormal sexual development in the fish.

WP020 (VAJ-1117-821646) Intersex and other forms of reproductive disruption in fishes downstram of a Colorado wastewater treatment plant effluent.
Start time: 8:00 AM
Vajda, A¹, Barber, L², Gray, J², Lopez, E¹, Woodling, J¹, Norris, D¹, ¹ University of Colorado, Boulder, Colorado, USA² U.S. Geological Survey, Boulder, CO, USA
Intersex white suckers (Catostomus commersoni) were collected on Boulder Creek downstream from wastewater treatment plant (WWTP) effluent and not from reference sites. This effluent is known to contain endocrine-active compounds including alkylphenol derivatives, bisphenol A, and reproductive steroids. The male to female ratio was skewed toward females at the downstream site in both spring and fall. Abnormalities in gonadal morphology, including smaller ovaries, less developed oocytes, and asynchronous follicular development were noted in adult female white suckers downstream of the WWTP. We also report elevated plasma vitellogenin in downstream juvenile white suckers. The reproductive potential of native fishes may be compromised in stream reaches of western states where large volumes of treated wastewater are discharged into relatively small-sized streams. Experiments exposing native fish species to treated wastewater will be discussed. This work was supported by a grant from the US EPA Region 8.

WP022 (BIS-1116-953708) The reproductive consequences of an environmentally relevant exposure of alkylphenol polyethoxylates on fathead minnow larvae.
Start time: 8:00 AM
Bistodeau, T¹, Cediel, R¹, Grove, K¹, Klaustermeier, J¹, Schoenfuss, H¹, ¹ St. Cloud State University, St. Cloud, MN, USA
Fathead minnow larvae (Pimephales promelas), less than 24 hrs old, were exposed for 64 days to a complex mixture of alkylphenol polyethoxylates which models the alkylphenol component of major metropolitan sewage treatment plant effluent. The exposure utilized a continuous flow-through system, designed to minimize contact with possible estrogenic surfaces. Water was supplied by a well and monitored throughout the exposure period. Water chemistry did not reveal any unusual conditions throughout the exposure. Following exposure, larvae were allowed to mature for four months in a similar flow-through, well supplied system. Upon sexual maturation, exposed male fish where allowed to compete with similarly reared, control males in a competitive spawning assay. Nest holding ability of control and exposed fish was carefully monitored for seven days. Here, the ecological significance of the effects of exposure to environmental estrogens can be clearly evaluated. All male fish were then sacrificed and analyzed for differences in the development of secondary sexual characters, developmental changes as measured by histology, and morphometric changes. Results indicate that exposure significantly reduces the ability of male fathead minnows to successfully hold and defend nest sites. Also, the expression of secondary sexual characters, GSI, and HIS were significantly reduced in exposed male fish. This is the first study in which larval fish were exposed during sexual differentiation to a mixture of alkylphenols identified in wastewater effluent.

WP023 (PHI-1117-635166) Concentrations and removal of pharmaceutical compounds at three wastewater plants in New York State, 2003-2004.
Start time: 8:00 AM
Phillips, P¹, Furlong, E², Stinson, B³, Zaugg, S², Esposito, K⁴, Anderson, J⁴, Kolpin, D⁵, ¹ US Geological Survey, Troy, NY, USA² US Geological Survey, Denver, CO, USA³ Metcalf and Eddy, Laurel, MD, USA⁴ Metalf and Eddy, New York, NY, USA⁵ US Geological Survey, Iowa City, IA, USA
Twelve influent and 12 effluent samples were collected during 2003-2004 at each of three wastewater-treatment plants in New York State; two use activated sludge (AS) and one uses a trickling filter (TF). The samples were analyzed for dissolved concentrations of 24 compounds by liquid chromatography/mass spectrometry. One or two samples from each site were collected as a 24 hour flow-weighted sample after biological treatment and filtering in addition to influent and effluent samples. Results indicate that 1) several pharmaceuticals are frequently present in influent entering all three plants, 2) chemical concentrations in influent differ among the plants, 3) the AS plants lowered the concentrations of many pharmaceuticals more than the TF plant, and 4) much of this decrease occurred during biological treatment. Thirteen of the pharmaceuticals were detected in more than 35 percent of the influent samples. Non-prescription compounds including acetaminophen, caffeine, and cotinine were routinely detected in influent at concentrations greater than 1 g/L, but most prescription compounds were detected at concentrations around 0.1 g/L. Median influent concentrations for acetaminophen, codeine, and fluoxetine (10, 10 and 100 g/L, respectively) at one AS plant were at least 10 times greater than those at the other two plants; this reflects differences in the sources. Comparison of influent concentrations with effluent concentrations indicates that concentrations of some compounds (including 1,7 dimethylxanthine, cotinine, acetaminophen, caffeine, and codeine) were decreased by 90 percent or more at AS plants and by 70 percent or less at the TF plant. Some compounds (including carbamazepine, dehydronifedipine and trimethoprim) were not removed by either type of plant. Analysis of samples collected before and after biological treatment, filtering, and disinfection, indicates that most of the concentration decrease occurs during biological treatment and that the filtration and disinfection processes have little effect on concentrations of most pharmaceuticals.

WP025 (MAR-1117-832582) Daily fluctuation in the estrogenicity of effluent released by a Great Lakes sewage treatment plant.
Start time: 8:00 AM
Martinovic, D¹, Sorensen, P¹, ¹ Dept. of Fisheries, Wildlife and Conservation Biology, University of Minnesota, St. Paul, MN, USA
Although it is well established that most sewage treatment plants release environmental estrogens (EE) and that the rates at which these compounds are released vary on a seasonal basis, fine-scale variation has not been examined. This is of concern because fathead minnows exposed to fluctuating levels of estrogen in the laboratory have been found to experience higher levels of vitellogenin expression than expected. This study examined daily variation in the estrogenicity of effluent discharged by the Western Lake Superior Sanitary District Plant (WLSSDP) in Duluth, Minnesota. This modern plant treats a combination of paper mill waste and domestic sewage. We sampled its effluent on a daily basis for three weeks in August 2003 and then analyzed its estrogenicity using an in vitro rainbow trout estrogen receptor binding assay (rtERA) while simultaneously examining liver vitellogenin mRNA expression in fathead minnows exposed to the same effluent. As measured by our rtER assay, the estrogenicity of WLSSDP effluent was high (mean of 42 ng/L estrogen equivalents (EEQ)) and variable (standard deviation of 25.4 ng/L EEQ). Because inter-sample variation was small in our rtER assay (standard deviation of 3.3 ng/L EEQ), and levels of mRNA expression showed similar trends in both magnitude and variability, we conclude that the changes we measured reflected biologically-relevant fluctuations in effluent estrogenicity. We are presently investigating the significance of analogous fluctuations in EE exposure on the reproductive success of fathead minnows in the laboratory. (Funded by Minnesota Sea Grant).

WP026 (BAL-1117-741427) Determination of sulfonamide antibiotics in wastewater: Comparing solid phase microextraction and solid phase extraction techniques.
Start time: 8:00 AM
Balakrishnan, Vimal¹, Terry, Ken¹, ¹ National Water Research Institute (Environment Canada), Burlington, ON, Canada
In recent years, pharmaceutical and personal care products (PPCPs) have been detected in diverse environments (including groundwater, river water, and municipal wastewater). In order to evaluate their environmental impact, PPCPs must first be accurately determined in environmental samples. In this study, we developed a LC-MS/MS method for the separation of 10 sulfonamide antibiotics in water: sulfaguanidine (SG), sulfacetamide (SAc), sulfadiazine (SD), sulfathiazole (ST), sulfapyridine (SP), sulfamerazine (SAm), sulfamethazine (SM), sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfasalazine (SS).While sulfonamides can easily be determined in pure water, more complicated matrices such as wastewater generate confounding matrix effects. For example, upon spiking wastewater samples obtained from water treatment plants in 3 Ontario municipalities with all 10 antibiotics, we found that SAc was subject to matrix suppression, while SG, SD, SAm, SM, and SS underwent matrix enhancement. Furthermore, LC-MS responses to ST, SP, SMX, and SDM were unaffected by the matrix. In an effort to overcome the observed matrix effects, we investigated two extraction techniques. 1) Solid Phase Extraction (SPE) on a variety of sorbent phases. We found that sequentially passing the samples through 1.2 m and 0.45 m filters followed by SPE on MCX cartridges yielded the greatest analyte recovery, and also succeeded in reducing matrix effects. 2) We then developed a Solid Phase Microextraction (SPME) method to determine sulfonamides, and applied it to wastewater samples. Of the 5 different fiber assemblies investigated, the Carbowax/Divinylbenzene (CW/DVB) fiber produced the optimal response to sulfonamides. We further optimized the SPME method according to: a) sorption time; b) desorption time; and c) salinity. In this presentation, the performance obtained for each compound by SPME will be compared to that obtained by SPE, and the strengths and limitations of each method in overcoming the matrix effects in wastewater will be discussed.

WP027 (AAA-1117-658057) Occurrence and Removal of Selected Pharmaceuticals and Personal Care Products (PPCPs) in Sewage Treatment Plants.
Start time: 8:00 AM
Yu, J¹, Bisceglia, K¹, Coelhan, M², Roberts, A¹, Bouwer, E¹, ¹ Johns Hopkins University, Baltimore, Maryland, USA² Technical University of Munich, Munich, Germany
In order to monitor the occurrence of pharmaceuticals and personal care products (PPCPs) in complex matrices such as wastewater influents and effluents, robust, accurate and highly reproducible analytical methods are required. We have developed two such methods to facilitate our study of the occurrence and removal of 53 PPCPs in sewage treatment plants (STPs). Both employ solid-phase extraction and chemical derivatization, either silylation using N, N-bis(trimethylsilyl)trifluoroacetamide/chlorotrimethylsilane, or alkylation with pentafluorobenzylbromide, followed in each case by analysis via gas chromatography/mass spectrometry. The target analytes span a range of therapeutic classes and commonly used personal care products. Twenty four of the 53 target analytes do not appear to have been previously sought in the United States, and many do not appear to have been previously studied. The occurrence and removal of these PPCPs has been investigated in five different STPs that employ different treatment processes and operational characteristics (e.g., different solid residence times and different nutrient removal processes). These treatment plants serve five different major cities within the mid-Atlantic and New England regions of the United States. Quadruplicate 24-hour composite influent and effluent samples were obtained at each site, and EPA method 526 guidelines were followed for quality assurance and quality control in analyzing wastewater samples. Many of the target analytes proved ubiquitous as micropollutants in both STP influents and effluents. Influent concentrations of PPCPs varied between geographic locations, and their removal efficiencies varied depending on the treatment processes and operational characteristics. Overall, the majority of the target analytes were detected in both the influent and effluent STP streams at ng/L levels, although some PPCPs (e.g., naproxen and ibuprofen) were encountered at g/L levels.

WP028 (HEM-1117-829567) Assessing removal of endocrine disrupting chemicals and activity during wastewater treatment.
Start time: 8:00 AM
Hemming, J¹, Drewes, J², Ladenburger, S², Mieritz, M¹, Pieters, R¹, Barman, M¹, Schauer, J¹, Sonzogni, W¹, ¹ Wisconsin State Laboratory of Hygiene, Madison, WI, USA² Colorado School of Mines, Golden, CO, USA
Using a combination of bioassays and chemical analytical techniques is important when assessing the removal of endocrine disrupting chemicals throughout wastewater treatment to determine which compounds are present and responsible for biological activity. Samples were collected from twelve U.S. water reclamation facilities and concentrated with solid phase extraction. GC-NCI-MS was used to quantify several steroidal hormones (testosterone, 17-estradiol, 17-ethinylestradiol, estrone, estriol) and phenolic compounds (bisphenol A, 4-tert-octylphenol, nonylphenol). Additionally, ELISAs after HPLC clean-up were used to determine testosterone, 17-estradiol and estriol concentrations. To evaluate estrogenic activity, we used the MCF-7 cell proliferation assay (E-screen) and an estrogen receptor transfected yeast assay. An androgen receptor (AR) transfected MCF-7 cell proliferation assay (A-screen) and an AR transfected yeast assay were used to assess androgen activity. The results of these assays, expressed in estrogen or androgen equivalents, agree reasonably well in assessing the general removal efficiencies of the treatment plant operations. Secondary treatment employing nitrification / denitrification more efficiently removed steroidal hormones than facilities employing no nitrification. Facilities employing long HRTs during secondary treatment exhibited hormone concentrations near or below the analytical detection limits. Membrane bioreactors removed compounds to a greater degree than secondary treatment, although not significantly so. UV disinfection treatment did not result in changes in bioactivity or hormone concentrations. Secondary and tertiary treatment followed by soil-aquifer treatment or advanced membrane treatment (microfiltration/reverse osmosis) seemed to efficiently remove endocrine disrupting activity. Differences in the potency of estriol in the two estrogen bioassays likely explain some of the differences in the activity between the two bioassays. Testosterone concentrations only account for a small portion of the androgenic activity. The androsterone found in some of the influents may account for some of the unexplained activity.

WP029 (ESP-1117-806919) Fate of estrogenic hormones and alkylphenols in representative French urban wastewater plants.
Start time: 8:00 AM
Bruchet, A.¹, Esperanza, M.¹, Gogot, C.¹, Huyard, A.¹, Janex-Habibi, M.¹, Herry, G², Raulet, C.², Mivelaz, P.², Meinhold, J.³, Castillo, L.³, ¹ CIRSEE. Suez Environnement, Le Pecq, France² Centre D'Analyses Environnementales. Veolia Environnement, Saint Maurice, France³ Anjour Recherche. Veolia Environnement., Maisons-Laffite, France
Urban wastewater plant effluents are one of the main inputs of endocrine disrupting chemicals (EDCs) in inland surface waters. There is now a consensus that most of the estrogenic effects observed in effluents can be attributed to natural and synthetic hormones excreted by humans. Alkylphenol derivatives, which have a much lower estrogenic activity but are present at much higher levels, also contribute to a lesser extent. An increasing number of papers are reporting the efficiency of wastewater plants for the removal of these EDCs from the liquid effluent, however little is known concerning the detailed fate of EDCs in the various treatment units of a wastewater plant.This paper will first describe the implementation of an integrated analytical scheme capable of measuring conjugated and deconjugated hormones, alkylphenols and alkylphenolethoxylates in both liquid (influent and effluent) and solid phases (suspended solids, different types of sludges). The four following hormones were targeted: b and a-estradiol, estrone and ethinylestradiol. The LC-MS/MS analytical approach comprised different extraction procedures but common clean-up steps. The methods for hormones were validated as part of an international interlaboratory exercise. Alkylphenols (both nonyl, octyl and their ethoxylates) were measured by GC/MS after silylation with MSTFA.A preliminary screening of 14 French domestic raw wastewater (from 5,000 to 350,000 IE) indicated the distribution and the range of concentrations encountered. These were rather homogenous for hormones but varied widely for alkylphenols. It also showed that conjugated hormones and hormones bound to suspended solids could not be neglected as they could represent up to 25% and 46% of the total influent load, respectively. Alkylphenols were mostly detected in the suspended matter.The analytical methods have been used to perform a mass balance calculation around different treatment units in wastewater treatment plant. This paper will report data obtained during the pre-screening and the mass balance approach.