Pharmaceuticals in Surface Waters

Transcription

1 Pharmaceuticals in Surface Waters Are Fish on Our Drugs? Marsha C. Black, PhD College of Public Health, University of Georgia February 20, 2010

2 Pharmaceuticals Many chemical types, MOA Antibiotics, analgesics, hormones, anti-epileptics, antidepressants, etc. Major forms Human prescription and over-the-counter drugs Veterinary drugs Parent compound, metabolites

3 Contamination of Surface Waters by Pharmaceuticals Mid-1970s Clofibric acid detected in European surface waters Recent improvements in analytical techniques Potential for effects on aquatic organisms?

4 Sources of Surface Water Contamination by Pharmaceuticals Human Drugs Disposal Landfill Livestock operations Metabolism, Excretion WWTP Groundwater Surface Waters Drinking Water

5 Wastewater Treatment Process 1º treatment: Remove solids 2º treatment: Decompose organics Activated sludge or trickling filter 3º treatment: Disinfection May also remove nutrients Discharge to surface water Are pharmaceutical chemicals removed?

6 Removal of Drugs by WWTP % 15% % 34% 15% Raw Sewage Effluent-TF % 75% 48% 69% 71% 83% 78% Effluent-AS 0 Bezafibrate Clofibric acid Ibuprofen Ketoprofen Indomethacin Naproxin Adapted from Stumpf, 1999

7 PPCP Presence in Aquatic Environments Clofibric acid detected in the North Sea, Swiss lakes (ng/l levels) Clofibric acid detected in German surface waters; Berlin drinking water Antibiotics detected in German surface waters Lipid regulators, anti-inflammatory drugs in WWTP effluents and river water in Rio de Janeiro, Brazil Beta-adrenergic receptor blocking drugs in WWTP effluent (US) Synthetic estrogens in urbanized, embayed marine environments Fluoxetine, sertraline and metabolites measured in fish collected from effluent-dominated stream (TX) Fluoxetine, sertraline detected in MS stream above/below WWTP Naproxin (ng/l) detected in drinking water in LA ETC

8 USGS Study ( ) Kolpin et al., Environmental Science and Technology 36: Sampled 139 streams in US Targeted susceptible locations Downstream from WWTPs, livestock operations Extracted, identified 95 analytes

9 USGS Reconnaissance Study Kolpin et al., Environmental Science and Technology 36:

10 Percent of total measured concentration of OWC by use category (Kolpin et al., 2002)

11 Results of USGS Survey (Kolpin et al., 2002) Low concentrations of PPCPs in streams Mostly < 1 ppb Toxicity to nontarget organisms largely unknown

12 Chemicals Detected in DW Sources Stackelberg et al., (2004) Science of the Total Environment 329:99-113

13 Chemicals Detected in Drinking Water Stackelberg et al., (2004) Science of the Total Environment 329: Raw water Settled Filtered Finished

14 Pharmaceuticals are designed to have a biological effect Why Worry? Aquatic organisms are exposed throughout their development Potential for multigenerational exposure Little known about fate in the environment

15 Selective Serotonin Reuptake Inhibitors (SSRIs) Treatment of clinical depression Evidence of endocrine disruption Widely prescribed Environmental Implications µg/l fluoxetine detected in US waters (Kolpin et al. 2002) µg/l fluoxetine detected in Canadian waters (Metcalfe et al. 2003) Induced spawning in bivalves (Fong 1998)

16 SSRI Structures F 3 C O CHCH 2 CH 2 NHCH 3 F NH O O O Fluoxetine (Prozac ) CN Paroxetine (Paxil ) F O CH 2 CH 2 CH 2 N(CH 3 ) 2 Citalopram (Celexa ) Cl F 3 C CH 2 CH 2 CH 2 CH 2 OCH 3 Cl NHCH 3 N O CH 2CH 2 NH 2 Fluvoxamine (Luvox ) Sertraline (Zoloft )

17 Diagram by Jacob L. Driesen, Ph.D. sri_actions.htm MOA of SSRIs in Mammals

18 Physicochemical Properties of SSRIs (data from Kwon and Armbrust) Compound Log K OC b Photolysis t ½c (d) Citalopram Fluoxetine Fluvoxamine ; 29 Paroxetine Sertraline

19 Detection of SSRIs in the Environment Fluoxetine (FL) ppb FL detected in USGS reconnaissance study (Kolpin et al. 2002) ppb FL in Canadian surface water (Metcalfe et al. 2003) ppb FL ; ppb norfluoxetine in Mississippi surface water (Wook-Kwon and Armbrust, unpublished) Sertraline (SE) ppb in Mississippi sediments 0.04 ppb SE; ppb norsertraline in Mississippi surface water (Wook-Kwon and Armbrust, unpublished)

20 Research Approach Characterize toxicity with invertebrates and fish Ceriodaphnia dubia Mortality; reproduction Mosquito fish (Gambusia affinis) Mortality; development Examine MOA Xenopus laevis

21 Acute Toxicity (LC50) of SSRIs in Ceriodaphnia dubia SSRI LC50 (ppb) Citalopram (Celexa ) 3180 Fluvoxamine (Luvox ) 1260 Paroxetine (Paxil ) 590 Fluoxetine (Prozac ) 470 Sertraline (Zoloft ) 140 LC50 = Concentration that kills 50% of the exposed organisms

22 Number of offspring (% of control) Effect of Fluoxetine on C. dubia Reproduction Fluoxetine concentration (ppb) *

23 Acute, Chronic Toxicity of Fluoxetine to Mosquitofish 7-d acute tests Mortality (LC50) Behavior Western mosquitofish Gambusia affinis

24 Mortality (%) Effect of Fluoxetine on Gambusia affinis 7-d LC50 = 546 ppb ppb 600 ppb 6 ppb 0.6 ppb Control Day

25 Effects of Fluoxetine on Fish Behavior Observations during 7-d acute tests 0.5 ppb fluoxetine Uncoordinated swimming Lethargy, lack of response to stimuli Less aggression, interaction between individuals Feeding study following 7-d exposures Mosquito fish fed Ceriodaphnia dubia 50, 100 ppb fluoxetine Significantly longer feeding times

26 Effects of Fluoxetine on Fish Development 85-d exposure Indicators of reproductive maturity Males: gonopodium Females: black spot (transient indicator) 71 ppb fluoxetine delayed development Male Female Gonopodium Black Spot

27 Acute and Chronic Toxicity Results Organism SSRI Duration/Endpoint Toxicity Value C. dubia SE 48 h; Mortality (LC50) 120 ppb C. dubia FL 48 h; Mortality (LC50) 510 ppb Mosquito fish Mosquito fish Mosquito fish FL 7 d; Locomotion, orientation 0.5 ppb FL 7 d; Feeding behavior 50 ppb FL 7 d; Mortality (LC50) 546 ppb C. dubia SE 7 d; # of offspring 45 ppb C. dubia FL 7 d; # of offspring 447 ppb Mosquito fish FL 85 d; sexual maturation 71 ppb

28 African Clawed Frog (Xenopus laevis) Easy to breed in the lab Inject with HCG Tadpole to froglet in d Many measurable endpoints Mortality Deformities Time to metamorphosis Good amphibian model Worldwide amphibian declines

29 Why Study Frogs? Metamorphosis cued by thyroid hormones No thyroid -- Metamorphosis inhibited Exposure to chemicals that interfere with T 3,T 4 Delay or inhibit metamorphosis Time to metamorphosis, feeding affected by corticosteroids

30 SSRIs Hypothalamus CRF (+) TRH (+) SSRIs Pituitary TSH (+) ACTH (+) PRL (-) Thyroid hormones stimulate limb growth and tail resorption, and are considered the primary hormones that control metamorphosis in frogs. Thyroid Gland TH (+) SSRIs Interrenal Gland Corticosteroids (+) Corticosteriods can inhibit early tadpole growth, but accelerate metamorphosis at later stages of development. Prolactin inhibits tail resorption, but promotes androgeninduced sexual differentiation of the male larynx. METAMORPHOSIS

31 Experimental Design Fluoxetine, Sertraline exposures ppb Positive control Ammonium perchlorate Negative control Fed frog brittle (0.5-1 mg/d) 70 days

32 Analyses of Adverse Effects Bioaccumulation (44 d) Endpoints Forelimb emergence (44 d) Tail resorption Time to metamorphosis Growth Mass at metamorphosis

33 Accumulation of SSRIs by Tadpoles (44 d) Treatment Water - Nominal (ppb) Tadpoles - Wet wt. (ppb) Fluoxetine 0.1 < LOD 1.0 < LOD (±0.04) Sertraline 0.1 < LOD (±0.00) (± 0.12)

34 Time to Metamorphosis (days) Timing of Metamorphosis Normal Development 50 * * Fluoxetine (µg/l) Sertraline (µg/l)

35 Body Weight (grams dry weight) Growth -- Mass at Metamorphosis * * * * * Fluoxetine (µg/l) Sertraline (µg/l)

36 Summary of Results Tadpoles accumulated FL and SE Growth reduced in tadpoles exposed to FL, SE Reduced feeding observed Suppression of feeding? Ecologically significant endpoint Decreased time to metamorphosis (SE) Adaptive response to reduced food intake? Effects observed at environmentally relevant concentrations

37 SSRIs Hypothalamus CRF (+) TRH (+) SSRIs Pituitary TSH (+) ACTH (+) PRL (-) Thyroid hormones stimulate limb growth and tail resorption, and are considered the primary hormones that control metamorphosis in frogs. Thyroid Gland TH (+) SSRIs Interrenal Gland Corticosteroids (+) Corticosteriods can inhibit early tadpole growth, but accelerate metamorphosis at later stages of development. Prolactin inhibits tail resorption, but promotes androgeninduced sexual differentiation of the male larynx. METAMORPHOSIS

38 Ecotoxicity of SSRIs SSRIs are toxic to C. dubia, mosquito fish Mortality Behavioral responses Suppressed reproduction SSRIs are toxic to Xenopus laevis Reduced size at metamorphosis Accelerated time to metamorphosis Stimulation of ACTH produce more cortisol? Increased vulnerability to predation; reduced feeding, mating success

39 Acknowledgements EPA Star Grant Research partners Dr. Ted Henry (now at UT/University of Plymouth, UK) Emily Rogers (MS, Toxicology; PhD candidate at UT) Dr. Deanna E. Conners (now with Oregon EPH) Lab assistance Nicki Campbell, Samantha Burton Ben Hale, Miles Buzbee Chemical analyses Drs. Kevin Armbrust and Jeong-Wook Kwon Mississippi State Chemical Laboratory R829006