EndocrineEndocrine EffectsEffects ofof SelectiveSelective SerotoninSerotonin ReuptakeReuptake InhibitorsInhibitors ((SSRIsSSRIs)) onon AquaticAquatic OrganismsOrganisms Marsha C. Black & Emily D. Rogers University of Georgia Athens, Georgia, USA Theodore B. Henry University of Tennessee Knoxville, TN OutlineOutline ¾¾ SSRIsSSRIs -- MOAMOA andand clinicalclinical significancesignificance ¾¾ PresencePresence inin thethe environmentenvironment ¾¾ StudyStudy objectivesobjectives ¾¾ ResultsResults andand DiscussionDiscussion z AcuteAcute toxicitytoxicity ((macroinvertebratemacroinvertebrate,, fish)fish) z ChronicChronic effectseffects ((macroinvertebratemacroinvertebrate,, fish,fish, frog)frog) ¾¾ SummarySummary andand conclusionsconclusions ¾¾ FutureFuture researchresearch directionsdirections SelectiveSelective SerotoninSerotonin ReuptakeReuptake InhibitorsInhibitors ((SSRIsSSRIs)) ¾¾ TreatTreat clinicalclinical depression,depression, obsessiveobsessive-- compulsivecompulsive andand panicpanic disorders,disorders, PMS,PMS, etc.etc. ¾¾ ClinicalClinical MOA:MOA: blockblock serotoninserotonin reuptakereuptake ¾¾ Examples:Examples: z FluoxetineFluoxetine (Prozac(Prozac®® andand SarafemSarafem®®)) z SertralineSertraline (Zoloft(Zoloft®®)) z CitalopramCitalopram ((CelexaCelexa®® andand LexaproLexapro®®)) z FluvoxamineFluvoxamine ((LuvoxLuvox®®)) z ParoxetineParoxetine ((PaxilPaxil®®)) SSRI Structures O F3C OCHCH2CH2NHCH3 FNH O O ® Fluoxetine (Prozac®) CN Paroxetine (Paxil ) F O CH2CH2CH2N(CH3)2 Citalopram ® (Celexa ) Cl CH2CH2CH2CH2OCH3 Cl NHCH3 F3C N CH2CH2NH2 O Fluvoxamine (Luvox®) Sertraline (Zoloft®) SourcesSources ofof SurfaceSurface WaterWater ContaminationContamination byby HumanHuman PharmaceuticalsPharmaceuticals Pharmaceuticals Disposal Metabolism, Excretion Landfill WWTP Groundwater Surface Waters SSRIsSSRIs:: DetectionDetection inin thethe EnvironmentEnvironment z FluoxetineFluoxetine detecteddetected inin surfacesurface waterswaters • 0.012 ppb detected in USGS reconnaissance study (Kolpin et al. 2002) • 0.030-0.099 ppb in Canada (Metcalfe et al. 2003) • 0.031-0.076 ppb in Mississippi (Wook-Kwon and Armbrust, unpublished) z FluoxetineFluoxetine,, sertralinesertraline andand metabolitesmetabolites detecteddetected inin fishfish tissuestissues (Brooks et al., 2005) PhysicochemicalPhysicochemical PropertiesProperties ofof SSRIsSSRIs (data from Wook-Kwon and Armbrust) a b c Compound Log KOW Log KOC Photolysis t½ (d) Citalopram 1.39 5.63 39 Fluoxetine 1.22 4.65 122 Fluvoxamine 1.21 3.82 0.57; 29 Paroxetine 1.37 4.47 0.67 Sertraline 1.37 4.17 23 aMeasured on salt form bAverage calculated from experiments with 5 different soils and sediments c Average calculated from experiments with 2 different lake water samples WhyWhy Worry?Worry? ¾ PharmaceuticalsPharmaceuticals areare designeddesigned toto havehave aa therapeutictherapeutic (=biological)(=biological) effecteffect z Effects on non-target organisms are mostly unknown ¾ AquaticAquatic organismsorganisms areare exposedexposed throughoutthroughout theirtheir lifetimelifetime ¾ PotentialPotential forfor multigenerationalmultigenerational exposureexposure ¾ LittleLittle isis knownknown aboutabout persistence,persistence, fatefate ofof drugsdrugs inin thethe environmentenvironment ¾ SSRIsSSRIs knownknown toto promotepromote spawningspawning inin mollusksmollusks OverallOverall ResearchResearch PlanPlan…… ¾ DetermineDetermine environmentalenvironmental fatefate ofof SSRIsSSRIs z Techniques used for pesticide registration z Measure hydrolysis, photolysis, metabolism, etc. ¾ MeasureMeasure parentparent andand majormajor degradationdegradation productsproducts z Wastewater effluent z Downstream receiving water ¾ DetermineDetermine acute,acute, chronicchronic impactsimpacts toto aquaticaquatic organismsorganisms z Ceriodaphnia dubia (macroinvertebrate) z Gambusia affinis (Western mosquito fish) z Xenopus laevis (frog) ToxicityToxicity TestsTests ¾ TestTest organism:organism: CeriodaphniaCeriodaphnia dubiadubia ¾ AcuteAcute toxicitytoxicity (48(48 h)h) z Single compound exposures z Binary, quaternary mixture exposures z Mortality (LC50) as endpoint ¾ ChronicChronic toxicitytoxicity z 7 day mini-chronic test z Brood size, # broods as endpoints ¾ AllAll teststests followedfollowed USUS EPAEPA protocolsprotocols AcuteAcute ToxicityToxicity (LC50)(LC50) ofof SSRIsSSRIs SSRI LC50 ppba Citalopram (Celexa®) 3180 (220) Fluvoxamine (Luvox®) 1260 (830) Paroxetine (Paxil®) 470 (60) Fluoxetine (Prozac®) 590 (130) Sertraline (Zoloft®) 140 (20) aMean (± SD) of 3 tests Henry et al. 2004, Environ Toxicol Chem 23:2229-2233 ChronicChronic ToxicityToxicity ofof SSRIsSSRIs SSRI NOECa LOECa (ppb) (ppb) Citalopram (Celexa®) 800 4000 Fluvoxamine (Luvox®) 366 1466b Paroxetine (Paxil®) 220 440b Fluoxetine (Prozac®) 89 447b Sertraline (Zoloft®) 9 45 aTotal number of neonates produced over 7-8 d bNumber of broods also significantly reduced (Henry et al. 2004, Environ Toxicol Chem 23:2229-2233) MixtureMixture ToxicityToxicity (In(In preparation,preparation, HenryHenry andand Black)Black) Equitoxic Mixture Equal Concentration Mixture 100 100 80 80 60 60 Observed 40 40 % Mortality Model Observed 20 Model CA 20 Model IA 0 0 0 5 10 15 20 25 012345 µM SSRI (quaternary mixture) AcuteAcute ToxicityToxicity ofof FluoxetineFluoxetine toto WesternWestern MosquitofishMosquitofish ¾ 77--dd acuteacute teststests ¾ Endpoints:Endpoints: z Mortality (LC50) z Fish behavior Western mosquitofish Gambusia affinis AcuteAcute ToxicityToxicity ofof FluoxetineFluoxetine toto WesternWestern MosquitofishMosquitofish ¾¾ MortalityMortality z 77--dayday LC50LC50 == 614614 ppbppb ¾¾ BehavioralBehavioral effectseffects (0.6(0.6 andand 66 ppb)ppb) z UncoordinatedUncoordinated swimmingswimming z Lethargy,Lethargy, lacklack ofof responseresponse toto stimulistimuli z LessLess aggression,aggression, interactioninteraction betweenbetween individualsindividuals ChronicChronic ExposuresExposures inin OutdoorOutdoor MesocosmsMesocosms •110-L plastic tanks •50 fish/tank •85-d exposure •Water change 1x/wk ChronicChronic TestsTests (140(140 d)d) withwith MosquitofishMosquitofish z TimeTime toto reproductivereproductive maturitymaturity • Fully developed gonopodium (males) • Formation of black spot (females) z HistologicalHistological effectseffects onon gonads?gonads? Male Female Gonopodium Black Spot EffectEffect ofof FluoxetineFluoxetine onon MaleMale SexualSexual DevelopmentDevelopment 50 a i 6 ppb d 40 60 ppb o p Control o n o 30 G * h t i 20 w t * n e c r 10 e P 0 020406080100 Days of Exposure* *Fish were 39-d old at t=0 EffectEffect ofof FluoxetineFluoxetine onon FemaleFemale SexualSexual DevelopmentDevelopment t 50 o p Control S k 40 6 ppb c a l * 60 ppb B 30 h t i * w t n 20 e c r e 10 P 0 0 1020304050 Days of Exposure* *Fish were 39-d old at t=0 ResearchResearch withwith thethe AfricanAfrican ClawedClawed FrogFrog ((XenopusXenopus laevislaevis)) ¾ EasyEasy toto breedbreed inin thethe lablab z Inject with HCG ¾ TadpoleTadpole toto frogfrog inin 6060--7070 dd ¾ ManyMany measurablemeasurable endpointsendpoints z Mortality z Developmental malformations z Time to metamorphosis WhyWhy StudyStudy Frogs?Frogs? ¾ ThyroidThyroid hormoneshormones (T(T3,T,T4)) cuecue metamorphosismetamorphosis ¾ TadpolesTadpoles withwith nono thyroidthyroid –– metamorphosismetamorphosis inhibitedinhibited ¾ ExposureExposure toto chemicalschemicals thatthat reducereduce circulatingcirculating TT3 willwill delaydelay oror inhibitinhibit metamorphosismetamorphosis RegulationRegulation ofof ThyroidThyroid AxisAxis inin MammalsMammals Serotonin X X X Fluoxetine www.dpcweb.com/images/medicalconditions/thyroid/thyroid%20illustration.jpg ¾ Serotonin inhibits the release of TRH from the hypothalamus in rats z Mitsuma et al. 1983; Mitsuma et al. 1996 ¾ Fluoxetine reduces circulating T3 and T4; increases TSH z Golstein et al., 1983 DoesDoes FluoxetineFluoxetine InhibitInhibit FrogFrog Metamorphosis?Metamorphosis? ¾ ExposeExpose tadpolestadpoles fromfrom hatchhatch untiluntil metamorphosismetamorphosis z FluoxetineFluoxetine (FL):(FL): 0.059,0.059, 0.295,0.295, 2.95,2.95, 29.529.5 ppbppb (measured)(measured) z AmmoniumAmmonium perchlorateperchlorate (AP):(AP): 1010 ppbppb z ControlControl (clean(clean exposureexposure water)water) ¾ ObserveObserve dailydaily forfor limblimb developmentdevelopment untiluntil metamorphosismetamorphosis isis completecomplete EffectsEffects ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine (Xenopus)(Xenopus) Tadpoles at 57 d* ¾ DevelopmentalDevelopmental delaysdelays z Forelimb formation z Tail resorbtion ¾ IncreasedIncreased timetime toto metamorphosismetamorphosis ¾ MortalityMortality Control 38 ppb FL 9.5 ppb AP *Data from range-finder experiment. Similar effects at 29.5 ppb in 2nd experiment. EffectEffect ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine onon TimeTime toto MetamorphosisMetamorphosis s i * s o 35 h p r 30 mo a t 25 e M o 20 t me 15 i T n i 10 e s a 5 e r c n 0 I Control 0.059 0.295 2.95 29.5 % Fluoxetine Concentration (ppb) EffectEffect ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine onon MassMass atat MetamorphosisMetamorphosis 45 * * * 40 * 35 Mass 30 25 20 15 % Reduction in 10 5 0 Control 0.059 0.295 2.95 29.5 Fluoxetine Concentration (ppb) EffectsEffects ofof ChronicChronic ExposureExposure toto FluoxetineFluoxetine (Exp.(Exp. 2)2) ¾ LimbLimb malformationsmalformations z Primary rotation of hindlimbs z Micromelia of forelimbs z Dorsal flexure of the tail TimeTime toto OnsetOnset ofof MalformationsMalformations