700 (SIM-1117-827609) Polycyclic Musks are estrogen receptor modulators: confirmation in vitro and in vivo.
Start time: 1:50 PM
Simmons, D, Sherry, J, Trudeau, V, Balch, G¹, Metcalfe, C¹, ¹ Trent University
Polycyclic synthetic musks are ubiquitous, and have been found in numerous river, sewage, lake and other water samples. Due to their lipophilicity (logKows = 5.7-5.9), HHCB and AHTN tend to accumulate in biological tissues. Gatermann et al., 1999, determined tissue levels of these polycyclic musks in North American biota, where Canadian freshwater fish ranged from 25 to 100 ng/g lipid, or 3 to 9 mg/kg wet.w. (Chemosphere, 38(14):3431-3441.). There is additional evidence that polycyclic musks may be weak environmental estrogens. Schreurs et al. (2002) demonstrated that both HHCB and AHTN were selective estrogen receptor modulators (SERMs) in vitro, meaning that they can induce estrogenic and antiestrogenic activity. Our Data indicate that concentrations of polycyclic synthetic musks in the tissues of fish from the lower Great Lakes may exceed 1mg/kg; depending upon sampling location and fish species. A three-tiered decision tree-design was used for this study. The yeast estrogen screen assay was used to first chose the most stronges estrogen modulator and a dual luciferase reporter gene assay was employed in rainbow trout gonadal cells with the rtER to assess interactions with fish estrogen receptors. The results of these assays indicated that both musks were inhibitors at the ER, however, HHCB had stronger effects at the hER and was therefore chosen for tests at the rainbow trout estrogen receptor. As an evaluation of the in vivo sublethal responses of fish to synthetic musks, rainbow trout were injected intraperitoneally with the polycyclic musk compound HHCB alone and HHCB plus E2. Vitellogen concentrations were reduced when HHCB was injected with E2.

701 (NEN-1116-576071) Effects of Carbamazepine and Fluoxetine on aquatic invertebrates.
Start time: 2:10 PM
Nentwig, G.¹, Hecker, V.¹, Oetken, M.¹, Oehlmann, J.¹, ¹ Johann-Wolfgang-Goethe-University, Zoological Institute, Frankfurt/M., Germany
The purpose of this research project was to determine effects of pharmaceuticals on freshwater invertebrates, as these compounds are biologically highly active. We were especially interested whether effects would occur at concentrations at which pharmaceutical residues are found in the aquatic environment. As only few literature data for exposure via sediment are available, we took this exposure pathway into consideration as well as water exposure. In the study, we exposed three freshwater invertebrate species to a range of pharmaceuticals. For the sediment bioassays, we used the midge Chironomus riparius and the oligochaete Lumbriculus variegatus as test organisms. The exposure pathway via water was assessed with the freshwater mudsnail Potamopyrgus antipodarum. Additionally, we used the ciliates Blepharisma japonicum and Tetrahymena thermophila as model organisms in a protist food web, reflecting effects on destruents. In this presentation, the results for the antiepileptic drug carbamazepine and the antidepressant drug fluoxetine will be shown. Carbamazepine is ubiquitously present in the aquatic environment; fluoxetine detections in rivers and streams are actually increasing. The results show that pharmaceuticals may have considerable effects on aquatic invertebrates and can pose a risk for the survival of field populations even at environmentally relevant concentrations.

702 (BRI-1117-726542) Reproductive effects of the pharmaceutical fluoxetine on a native freshwater mussel.
Start time: 2:30 PM
Bringolf, R.¹, Cope, W.¹, Heltsley, R.², Eads, C.³, Shea, D.¹, ¹ Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, USA² Hollings Marine Laboratory, National Institute of Standards and Technology, Charleston, SC, USA³ College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA
Recently, widespread occurrence of fluoxetine and other pharmaceuticals and personal care products (PPCPs) has been reported in surface waters of the United States and elsewhere; however, impacts of PPCPs on aquatic organisms are largely unknown. Fluoxetine is the active ingredient in the prescription anti-depressant drug Prozac^TM, which acts as a selective serotonin reuptake inhibitor (SSRI) to increase serotonin levels at nerve synapses. Serotonin has previously been used to induce parturition/spawning in some bivalves. To determine the potential of fluoxetine to disrupt native freshwater mussel reproduction, we exposed gravid adult female eastern elliptio (Elliptio complanata) mussels to fluoxetine at target concentrations of 0, 0.03, 0.3, 3.0, 30, 300, and 3000 g/L for 96 h in static-renewal tests. We used serotonin as a positive control and methiothepin, an inhibitor of serotonin pathways, as a confirmatory agent of pathway effects. Time to glochidia release, glochidia maturity and viability, and the number of glochidia released (fecundity) were measured. Fluoxetine exposure concentrations were analyzed by liquid chromatography/mass spectroscopy (LC-MS) to confirm exposure concentrations. Time to glochidial release was variable among the fluoxetine treatment groups, but was generally inversely related to exposure concentration. No glochidia were released by mussels in the control group in < 72 h; however, some mussels in each fluoxetine treatment, including low, environmentally relevant concentrations, released glochidia in < 24 hours. The average number of glochidia released was variable, but was generally positively correlated with fluoxetine exposure concentration. Released glochidia were predominantly immature, indicating that fluoxetine induced parturition in gravid mussels. Implications for population-level effects will be discussed. Additional studies of the endocrine and reproductive effects of fluoxetine on ripe adult male mussels are needed.

703 (ROG-1117-829066) Effect of predator cues on native tadpoles (Hyla chrysoscelis) exposed to fluoxetine (Prozac^®).
Start time: 2:50 PM
Rogers, E¹, Maerz, J², Davis, A², Black, M¹, ¹ Interdisciplinary Toxicology Program, Department of Environmental Health Science, University of Georgia, Athens, GA, USA²
Fluoxetine (Prozac^®) has been detected at low concentrations in surface waters and has the potential to affect non-target aquatic species, including developing amphibians. In a previous study conducted in our laboratory, Xenopus laevis tadpoles exposed to environmentally relevant concentrations of fluoxetine (0.059 ppb) exhibited limb malformations and reductions in mass at metamorphosis. Metamorphosis was also delayed at a higher concentration (29.5 ppb); however, this concentration exceeds environmental levels. Because our study used an exotic species and tadpoles were exposed to fluoxetine individually in small aquaria, it is difficult to extrapolate these results to native tadpoles developing under natural conditions. The aim of the present experiment is to expose native tadpoles to fluoxetine under natural densities with the added stress of a predator. Studies have shown that the presence of a predator can reduce tadpole feeding and delay metamorphosis. Since fluoxetine has been shown to delay metamorphosis at high concentrations, we hypothesize that this effect may be seen at lower concentrations with the addition of a predator. Groups of 20 gray tree frog tadpoles (Hyla chrysoscelis) will be exposed in 50 L containers to 0 - 0.2 ppb fluoxetine (n = 8 per concentration). Half of the replicates will receive a caged dragonfly larva, while remaining replicates will receive an empty cage. Exposure will begin early in development and continue through metamorphosis and will take place in a greenhouse under natural temperature and photoperiod. Each week, a subset of tadpoles will be staged and weighed, and daily observations for forelimb emergence, tail resorbtion, survival and malformations will be conducted. Results will be compared across treatments to determine if fluoxetine alone or with predator addition will impair development. We predict that the additional stress of a predator will produce a greater response than fluoxetine alone.

(59262) break.
Start time: 3:10 PM

704 (MCD-1117-650854) The impact of chronic, waterborne selective serotonin reuptake inhibitor (SSRI) exposure on toadfish physiology.
Start time: 3:50 PM
McDonald, M.D.¹, Julian, C¹, Riemer, D.D.¹, ¹ RSMAS, University of Miami, Miami, Florida, USA
Pharmaceuticals that modulate serotonin (5-hydroxytryptamine; 5-HT) are amongst the most prescribed in the United States. After consumption, selective serotonin reuptake inhibitors (SSRIs), which cause an increase in local 5-HT concentrations, are extensively metabolized by the liver. Their metabolites as well as 10-15% of the unchanged parent drug are eliminated from the body via the renal or gastrointestinal systems. Many consumed pharmaceutical compounds, including SSRIs, enter sewage treatment facilities and are not degraded. Consequently, these compounds have reached measurable quantities in surface waters. The potential contamination of the Florida Keys region and the Florida Keys National Marine Sanctuary (FKNMS) by these compounds is currently under investigation and the impact of pharmaceutical exposure on marine life in the area needs to be thoroughly examined. The gulf toadfish, Opsanus beta, is a benthic, marine teleost fish abundant along the Florida coastline and within the FKNMS. Recent work on the toadfish has shown that the HPI axis, branchial nitrogen excretion, branchial blood flow and intestinal ionoregulatory processes are regulated by 5-HT and both 5-HT receptors and transporters have been identified pharmacologically in these organs. To test the chronic impact of two of the most prescribed selective serotonin reuptake inhibitors (SSRIs) on gulf toadfish physiology, fish were separated into five waterborne exposure groups: control, 0.01 g/l fluoxetine-HCl (environmentally relevant), 10 g/l fluoxetine-HCl, 0.01 g/l sertraline-HCl or 10 g/l sertraline-HCl); and were exposed at these nominal concentrations for 60 days. The concentration of SSRIs in the water and accumulation in toadfish tissues and fluids were analyzed by liquid chromatography (LC)/ electrospray/ion trap mass spectrometry (MS) (Agilent Technologies and Bruker Daltronics) after appropriate sample extraction. The impact of chronic SSRI exposure on acid/base balance, the HPI axis, ionoregulation and 5-HT-regulated processes including branchial nitrogen excretion and branchial vascular resistance will be determined both in vivo and in vitro using a suite of physiological techniques.

705 (OWE-1117-622554) Cardiac and Growth Performance of Rainbow Trout (Oncorhynchus mykiss) Exposed to Propranolol: Non-invasive Fish ECG.
Start time: 4:10 PM
Owen, S¹, Huggett, D², Hutchinson, T³, Kinter, L⁴, Ericson, J², Hetheridge, M³, Sumpter, J¹, ¹ Brunel University, London, UK² Pfizer, Global R&D, Groton, CT, USA³ AstraZeneca, Global Saftey Health and Environment, Brixham, UK⁴ AstraZeneca Pharmaceuticals, Wilmington, PE, USA
Propranolol, a non-specific beta1/beta2 adrenergic antagonist has been detected in sewage treatment effluents and receiving waters in Europe and USA. Rainbow trout have beta-adrenergic signalling systems analogous to human cardiovascular receptors that respond to pharmacological doses of agonists and antagonists. Additional beta-receptor sub-types associated with functions that are physiologically different to the known mammalian functions suggest propranolol may interact with non-cardiovascular systems such as those for homeostasis, reproduction and immunocompetence. Little information is available on the physiological effects of water-borne propranolol or plasma concentrations in exposed fish. We report that trout took propranolol up from water such that after 40 days of exposure/acclimation, the linear relationship between water and plasma concentrations was described by [plasma] = 0.59[water], (n=31, r=0.96). Thus, uptake may potentially be modelled in fish by accounting for lipophillicity and processes such as protein binding and metabolism of the pharmaceutical. Physiological and biometric effects were also examined. Growth-rate was affected only at very high propranolol concentrations (LOEC 10mg/l). However, additional endpoints suggested morphological responses on the liver and heart size at 100ng/l (close to environmental measured range), the consequences of which are unknown at present. Interestingly, the 'mammalian-fish leverage model' (Huggett et al., 2004) predicts plasma concentrations at these exposures that would fall within the range of active mammalian plasma concentrations. Additional endpoints were examined including ECGs from unrestrained fish. In this presentation we report a preliminary study using waterborne cardio-modulators (ethanol and propranolol) and using spectral analysis of the ECG to examine heart rate variability, changes in QRS complex and QT-period in comparison with other measures of overall performance and homeostasis such as growth and condition.

706 (KID-1117-827167) Responses of a freshwater food web to a potent estrogen.
Start time: 4:30 PM
Kidd, K^{1, 2}, Paterson, M.², Blanchfield, P.², Mills, K.², Findlay, D.², Salki, A.², ¹ University of New Brunswick, Saint John, New Brunswick, Canada² Fisheries and Oceans Canada, Winnipeg, Manitoba, Canada
A whole-lake study was conducted from 1999-2005 at the Experimental Lakes Area in northwestern Ontario to determine whether the potent synthetic estrogen 17 ethynylestradiol (EE2) affects freshwater food webs. EE2 was added continuously to Lake 260 in each of the summers of 2001-2003 to maintain surface water concentrations between 4 and 6 ng L^-1. Water chemistry and microbial, algal, and zooplankton populations were sampled in this lake bi-weekly in the two years before and after dosing and in the three years of EE2 additions, and compared to reference lake samples collected over the same time period. In addition, large and small fish species were monitored for population-level effects in the spring and fall of each year in the EE2 amended and reference lakes. Microbial abundances ranged from 0.04 to 0.3 mg L^-1 in the study lake, and were similar before and after the additions of the estrogen. In contrast, the algal biomass in Lake 260 was lowest in 2003 at 465 mg m^-3 compared to 600-900 mg m^-3 in previous years, and may be related to increased grazing due to changes in food web structure. Mean annual zooplankton biomass in the study lake was similar from 1999-2003 and ranged between 20 to 80 ug L^-1. In 2003, however, there was increased abundance of Daphnia pulex, Diaphanosoma birgei, and rotifers when compared to previous years' data. This change in the zooplankton community composition may be due to the effects of EE2 on the fish species in Lake 260. As one example, a loss of the smaller size classes was observed for the fathead minnow population. Results from this study suggest that a potent estrogen can have both direct and indirect effects on aquatic populations.

707 (SOL-1117-854036) Risk assessment of veterinary pharmaceuticals in surface waters of a watershed in Southern Ontario, Canada.
Start time: 4:50 PM
Solomon, K¹, Lissemore, L¹, Hao, C², Yang, P², Mabury, S³, ¹ University of Guelph, Guelph, ON, Canada² Ontario Ministry of Environment, Toronto, ON, Canada³ University of Toronto, Toronto, ON, Canada
Few data are available that characterize the sources, exposure and effects of pharmaceuticals in the environment and there is clearly a need to define these parameters within a Canadian context. Of the twenty-nine pharmaceuticals surveyed in a model watershed in S. Ontario, 14 were detected in surface waters. Although not used in agricultural situations, carbamazapine was detected in strictly agricultural watersheds without WWTP inputs. The two most frequently detected antibiotics were lincomycin and monensin. Temporal trends in concentration for 5 frequently detected pharmaceuticals showed pulses occurring between May and November of 2003 at similar but varying times over the seasons, depending on the pharmaceuticals, flow rate, and precipitation. Fluctuations in concentration of ions indicative of agricultural run off, such as nitrate and phosphate, were not found to be useful predictors of changes in pharmaceutical concentration (r² < 0.4). When compared to acute toxicity values or QSAR estimates, distributions of estimated total potency of pharmaceuticals detected in the surface waters suggested small risks from acute effects of mixtures to daphnia, green algae, Lemna gibba, and fish. Microtox assays conducted on extracts of surface waters showed greater toxicity than could be explained by the total potency of the pharmaceuticals detected in the samples. This work was supported by the Canadian Network of Toxicology Centers, Agriculture Canada Livestock Environmental Initiative, and The Beef Cattle Research Council.

708 (CUN-1117-823257) Human Health and Environmental Risk Assessment of Carbamazepine in Surface Waters of the United States and Europe.
Start time: 5:10 PM
Cunningham, V.¹, D'Aco, V.², Bechter, R.³, Groff, J.⁴, Hartmann, A.³, ¹ GlaxoSmithKline, King of Prussia, PA, USA² Quantum Management Group, Inc., Clifton, NJ, USA³ Novartis Pharma AG, Basel, Switzerland⁴ Merck & Co., Inc., Elkton, VA, USA
Carbamazepine is a pharmaceutical compound approved worldwide as a medicine for treating epileptic seizures and trigeminal neuralgia. It is also sometimes used to treat bipolar depression, excited psychosis, and mania. Since 1998, it has been reported as detected in surface waters in Europe, the United States and Canada. Carbamazepine tends to be detected more frequently and at relatively higher concentrations than most other active pharmaceutical compounds (APIs). In addition, studies of its fate in wastewater treatment systems and surface waters suggest that it may be more resistant to degradation than other APIs. This recalcitrance and its relatively high prescription volume may explain, at least in part, the greater detection frequency of carbamazepine in the environment. There have been a relatively large number of papers published on its aquatic fate and effects (more than 20 publications, most published since 2002). This paper summarizes all the peer-reviewed information available to date on the toxicology and aquatic toxicity of carbamazepine, develops surface water concentrations considered protective of human health and both acute and chronic exposures to aquatic life, and compares those concentrations to both measured and modeled concentrations for the United States and Europe.