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Waterborne and sediment toxicity of fluoxetine to select organisms Bryan W. Brooks1,2, Philip K. Turner1, Jacob K. Stanley1, James J. Weston3, Elizabeth A. Glidewell1,2, Christy M. Foran4, Marc Slattery3, Duane B. Huggett3 1Institute of Applied Sciences, Department of Biological Sciences, University of North Texas, Denton TX 2Current Address: Department of Environmental Studies, Baylor University, Waco TX 3Environmental Toxicology Research Program, The University of Mississippi, University MS 4Department of Biology, West Virginia University, Morgantown, WV.
_ P. promelas eggs were acquired from breeding cultures maintained at the Institute of Applied Sciences, Denton, TX.
Ecological risk assessments of pharmaceuticals are currently difficult because little to no aquatic hazard and exposure information exists in the peer-reviewed literature for most therapeutics. Recently we identified _ Prior to toxicity testing, juvenile fish were fed 24-48 h old Artemia nauplii twice daily.
fluoxetine, a widely prescribed antidepressant, in municipal effluents. To assess potential hazard of _ Two 48-hour acute toxicity tests were conducted with 11- and 14-day old fathead minnows, respectively (10).
fluoxetine to aquatic biota, standardized, single species laboratory toxicity tests were performed. Average _ Water quality parameters for reconstituted hard water and balanced saline salt solution are listed in Figure 1. Effects of fluoxetine on C. dubia reproduction Figure 2. Effects of fluoxetine on H. azteca reproduction LC values for Ceriodaphnia dubia, Daphnia magna, and Pimephales promelas were 0.756, 2.65, and 2.28 _ Organisms were not fed during 48-hour tests. A light cycle of 16 h light, 8 h dark was maintained at 25±1°C µM, respectively. Pseudokirchneriella subcapitata growth and C. dubia fecundity were affected by 0.044 _ Sediment total organic carbon was 22340 mg/kg; percent moisture was 60%. Sediment grain size was and 0.72 µM fluoxetine treatments, respectively. Oryzias latipes survival was not affected by fluoxetine distributed as 41.2% sand, 39.2% silt and 19.6% clay.
exposure up to a concentration of 28.9 mM. In addition, 10-day sediment fluoxetine exposures were _ Percent fluoxetine recoveries from aquatic test concentrations ranged from 95 to 113%; therefore, Oryzias latipes were cultured according to methods of Yamamoto (11).
conducted with Chironomus tentans and Hyalella azteca, and a 42-day study was performed to evaluate nominal concentrations are presented in Figures 1-3 and Table 2.
fluoxetine effects on H. azteca reproduction. An LC value of 17 mg/kg was estimated for C. tentans; _ Ten organisms each were placed in three replicate beakers at nominal concentrations of 0, 1.8, 3.6, 7.2, 14.5, and 28.9 mM however, H. azteca survival was not affected by a 50 mg/kg fluoxetine sediment exposure level. H. azteca and C. tentans growth LOECs were determined to be 6 and 1 mg/kg, respectively. Our findings indicate _ Temperature was maintained at 25±1°C with a 16 h light, 8 h dark cycle.
that lowest measured aqueous fluoxetine effect levels are one to two orders of magnitude higher than the highest fluoxetine measures in municipal effluents.
_ O. latipes and H. azteca survival was unaffected across treatment levels. An LC of 15.2 mg/kg was estimated for C. tentans (Table 2). Average LC values for C. dubia, D. magna, and P.
promelas were 0.756, 2.65, and 2.28 µM (Table 2), respectively.
Sediment toxicity tests were performed using a Zumwalt testing system (12).
_ Fluoxetine LOEC and NOEC levels on C. dubia reproduction were 360 and 180 nM, respectively _ Dechlorinated, activated carbon treated tap water served as overlying water (12).
(Figure 1). The mean difference between 360 nM and 0 treatments was just 2.1 neonates.
_ C. tentans growth was reduced at each level, with a LOEC of 1.3 mg/kg (the lowest treatment level).
_ Reference sediments were obtained from pond mesocosms at the University of North Texas Water Research Field Station.
H. azteca growth was also significantly reduced at all levels, with a LOEC of 5.6 mg/kg (also the Physical sediment parameters were evaluated including organic carbon, particle characterization, and percent water.
_ Recent research indicates that multiple classes of pharmaceutical chemicals are present in municipal _ Greater C. tentans sensitivity to fluoxetine than H. azteca may result from increased exposure via wastewater effluents and surface waters (1-3).
_ Sediments were spiked with fluoxetine according to methods of Suedel and Rodgers (13).
_ Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is a commonly prescribed antidepressant _ In a 42-day study, H. azteca fecundity was not significantly affected by fluoxetine treatments (Figure 2).
(4) also found in effluents and surface waters (2,5).
Chironomus tentans and Hyalella azteca All treatment levels appeared to stimulate H. azteca reproduction (Figure 2).
_ Few studies have evaluated environmental effects of fluoxetine (6-7; also see Richards et al., Weston _ An increase in C. dubia fecundity at 180 nM was also observed (Figure 1).
10-day C. tentans and H. azteca, and 42-day H. azteca toxicity tests followed standard methods (12).
et al., Slattery et al. this meeting).
_ P. subcapitata growth responses to fluoxetine were evaluated by cell density and turbidity with _ Following preliminary range finding toxicity tests, 10-day C. tentans and H. azteca treatment levels were selected at 0, 1.4, Figure 3. Pseudokirchneriella subcapitata growth responses to fluoxetine estimated EC values of 126 nM by cell density and 77 nM by turbidity (Table 2). Growth (by The primary objective of this study was to evaluate the environmental hazard of fluoxetine to select 2.8, 5.6, 11.2 and 22.4 mg/kg, and 0, 5.4, 10.8, 21.6 and 43.2 mg/kg, respectively.
turbidity) was significantly reduced at 43.6 nM and at 174.4 nM (by cell number) (Figure 3).
benthic and pelagic toxicity test organisms.
_ Cell deformities were observed and cell sizes appeared smaller at 87.3 and 174.4 nM treatment levels.
Treatment levels for the 42-day H. azteca reproduction study were selected at 1.4, 2.8, 5.6, 11.2, and 22.4 mg/kg.
_ This study provides fluoxetine aqueous toxicity data for Pseudokirchneriella subcapitata (formerly Cells appeared shriveled and occasionally were not crescent shaped, a normal characteristic of P.
Selanastrum capricornutum), Ceriodaphnia dubia, Daphnia magna, Pimephales promelas and Oryzias latipes, and sediment toxicity data for Hyalella azteca and Chironomus tentans.
The mechanism of toxicity for observed cell deformities is not known; however, fluoxetine is known to have bacteriostatic effects, perhaps exerted by efflux pump inhibition (15). Although cell _ C. dubia, D. magna, and P. promelas bioassays were performed in reconstituted hard water (RHW).
deformities and biovolumes were not quantified in this study, such an effect of fluoxetine on algal cell wall structure warrants further investigation.
_ O. latipes tests were performed in a balanced saline salt (BSS) solution (11).
For each study, temperature, pH, dissolved oxygen, conductivity or salinity, alkalinity and hardness measures followed According to Weston, et al. (5), 100 mL samples of treatment levels were adjusted to pH=9, prefiltered with 1.2 _m GFF, and extracted with hexane and methanol conditioned Empore® C18 solid phase extraction disks. Disks were eluted with _ Sensitivities of organisms tested in this study to fluoxetine were P. subcapitata > C. dubia > Algal tests followed general procedures recommended by the US EPA (8).
methanol, brought to dryness, resolublized in 100 _L methanol, and analyzed by a Waters Alliance model 2690 liquid P. promelas > D. magna > C. tentans > H. azteca.
chromatograph with Waters 996 photo diode array and Waters Micromass ZQ mass selective detectors. Method detection _ Fluoxetine was added to three replicate 125-ml flasks at 0, 43.6, 87.3, and 174.4 nM. 25 ml sterile AAP _ Weston, et al. (5) found municipal effluent fluoxetine concentrations to range from 0.3 to 1.6 nM; limits were 0.2 ng/L. Sediment fluoxetine concentrations were not verified.
media was inoculated with 4 to 7-day-old stock culture algae such that flasks contained this range is higher than levels reported by Kolpin et al. (2). The lowest fluoxetine effect approximately 1 × 104 cells/ml at test initiation.
level reported in this study was 43.6 nM for algal growth. Based on effects data presented here, _ Flasks were placed under cool white fluorescent lighting (2.15 X 103 lm*m-2) with a light cycle of 16 h a Hazard Quotient is calculated at < 1 (7).
light, 8 h dark. During a 120 h exposure period, temperature was maintained at 25±1°C and _ Our results indicate that fluoxetine adversely affects aquatic organisms at levels one to two orders of flasks were hand-swirled twice daily.
_ Pseudokirchneriella subcapitata EC levels were estimated using nonlinear regression with the SAS system (14).
magnitude higher than the highest reported municipal effluent concentrations.
_ Algal growth was evaluated by enumeration and turbidity measurements (8). Cell counts were made _ LC values for C. dubia, D. magna, P. promelas and C. tentans tests were estimated using Trimmed Spearman Karber.
_ Based on data presented here, expected environmental fluoxetine concentrations are not acutely using a hemocytometer and a Nikon® compound microscope. Turbidity was determined as Table 2. P. subcapitata growth (EC50) and C. dubia, D. magna, P. promelas toxic to standard aquatic bioassay organisms.
absorbance with a Beckman DU 64 spectrophotometer at 750 nm.
_ P. subcapitata, H. azteca and C. tentans growth, and C. dubia and H. azteca fecundity responses were evaluated using a 50) responses to fluoxetine treatments.
Potential chronic responses of non-target biota, including behavioral responses and 5-HT levels, and one-way ANOVA with Dunnett’s test for multiple comparisons.
ecological communities require further investigation (7).
_ C. dubia and D. magna were mass cultured as previously described (9).
__________________________________________________________________________________ _ C. dubia and D. magna 48-hour acute toxicity tests were performed with less than 24-hour old neonates Table 1. Water quality characteristics of aqueous toxicity tests.
_ A seven day study evaluated C. dubia fecundity responses to fluoxetine treatments at nominal levels of ____________________________________________________ 0, 45.5, 91, 181, 362 and 724 nM (8).
1. Ternes T. 1998. Occurrence of drugs in German sewage treatment plants and rivers. Water Res. 32(11): 3245-3260.
_ Organisms were fed a 0.5 ml algae-Cerophyll suspension following daily renewals of test solutions (9).
2. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999-2000: A national reconnaissance. Environ. Sci. Tech. 36(6): 1202-1211.
____________________________________________________________________________ _ All studies were performed at 25±1°C with 16 h light, 8 h dark cycles.
3. Huggett DB, Brooks BW, Peterson B, Foran CM, Schlenk D. 2002. Toxicity of select beta-adrenergic receptor blocking pharmacueticals (β - blockers) on aquatic organisms. Arch. Environ. Contam. Toxicol. 43: 229-235.
4. rxList. 2002. http://www.rxlist.com/top200.htm.
5. Weston JJ, Huggett DB, Rimoldi J, Foran CM, Stattery M. 2001. Determination of fluoxetine (Prozac™) and norfluoxetine in the aquatic environment. Annual Meeting of the Society of Environmental Toxicology and Chemistry, Baltimore, MD.
6. Fong PP, 2001. Antidepressants in aquatic organisms: A wide range of effects. In: Daughton CG, Jones-Lepp TL, Eds. Pharmaceuticals and personal care products in the environment: Scientific and Regulatory Issues, American Chemical Society, Washington, DC.
_________________________________________________________ 7. Brooks BW, Foran CM, Richards SM, Weston J, Turner PK, Stanley JK, Solomon KR, Slattery M, La Point TW. 2003. Aquatic ecotoxicology of fluoxetine. Toxicol. Lett. Accepted.
8. USEPA. 1989. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms, 2nd LC values are averaged for duplicate experiments.
Edition. EPA 600/4-89-001. Environmental Monitoring Systems Laboratory, Cincinnati, OH.
This research was supported in part by a U.S. Congressional Signals and Sensors grant to M Slattery and 9. Hemming JM, Turner PK, Brooks BW, Waller WT, La Point TW. 2002. Assessment of toxicity reduction in wastewater effluent flowing through a treatment wetland using Pimephales promelas, Ceriodaphnia dubia, and Vibrio fischeri. Arch. Environ. Contam. Toxicol. 42: 9-16.
CM Foran, a Texas Water Resources Institute/United States Geological Survey grant to BW Brooks, the 10. USEPA. 1991. Methods for Measuring the Acute Toxicity of Effluents Receiving waters to Freshwater and Marine Organisms, 4th Edition.
Institute of Applied Sciences at the University of North Texas, and the Environmental Toxicology Research 11. Yamamoto TS. 1975. Medaka “kilifish”: Biology and strains. Keigaku Publishing Co., Tokyo.
Program at the University of Mississippi. The authors thank Dr. John Rimoldi, Erica March and Bethany 12. USEPA. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, 13. Suedel B, Rodgers Jr. JH. 1996. Toxicity of fluoranthene to Daphnia magna, Hyalella azteca, Chironomus tentans, and Stylaria lacustris in water-only and whole sediment exposures. Bull. Environ. Contam. Toxicol. 57: 132-138.
14. Bruce RD, Versteeg DJ. 1992. A statistical procedure for modeling continuous toxicity data. Environ. Toxicol. Chem. 11: 1485-1494.
___________________________________________________________________________ 15. Munoz-Bellido JL, Munoz-Criado S, Garcìa-Rodrìguez JA. 2000. Antimicrobial activity of psychotropic drugs: selective serotonin reuptake inhibitors. Inter. J. Antimicrob. Ag. 14: 177-180.

Source: http://riogrande.tamu.edu/reports/2002/2002-056/brooks-fluoxetine.pdf

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