A comparison of electronic and traditional cigarette butt leachate on the development of xenopus laevis embryos

A comparison of electronic and traditional cigarette butt leachate on the development of Xenopus laevis embryos F A t T a b a A R R 1 A A K C X D t e w U f i C C p w c i m t c h 2 Toxicology Reports 4[.]

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j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / t o x r e p

Full length article

Tatiana Tatum Parkera,∗, James Rayburnb

a Department of Biological Sciences, Saint Xavier University, 3700 West 103 rd St., Chicago, IL, 60655, United States

b Department of Biology, Jacksonville State University, 700 Pelham Rd N, Jacksonville, AL, 36265, United States

a r t i c l e i n f o

Article history:

Received 19 August 2016

Received in revised form

16 December 2016

Accepted 23 January 2017

Available online 24 January 2017

Keywords:

Cigarette butt leachate

Xenopus laevis

Development

a b s t r a c t

Cigarettebuttsare,collectively,themostcommonformof

lit-terintheworld.Approximately5.6trillioncigarettesaresmoked

everyyearworldwide.IntheUnitedStatesofAmerica,cigarette

wasteconstitutesanestimated30%ofthetotallitter(bycount)on

U.S.shorelines,inwaterways,andonland[1].Thecurrentmethod

formeasuringofhowmanycigarettebuttsarefindingtheirway

intostreams,rivers,andcoastalenvironmentsistheInternational

CoastalCleanupDay,whichis organizedannuallybytheOcean

Conservancy.Theevent involvesmorethan 500,000volunteers

pickingupdebrisfrombeaches,rivers,and streamsaroundthe

world.ThevolunteerscompleteMarineDebrisDataCardsthat

indi-catethequantityandtypeoflittertheypickup.Duringthe2013

internationalcleanup, 2,043,470 cigarette butts were collected,

makingthisthemostcommondebrisitem.Cigarettebuttshave

toppedthelistinallInternationalCoastalCleanupDayssincethat

categorywasaddedtothedatacardsasaseparateitemin1990[2]

∗ Corresponding author.

E-mail address: tatum@sxu.edu (T.T Parker).

Thefiltersinonepackof20cigarettesweigh0.12ounces(with

notobaccoattached)and displacea volumeof10mL.Based on thesefigures,thecigarettebuttscollectedin2013weighed approx-imately 766lbs and displaceda volumeof1022l.Owingtothe ubiquitous natureand magnitude of cigarette butts discharged intotheenvironment,studieshavebeenundertakentodetermine whethercigarettebuttwastecanexertecotoxiceffectsinaquatic environments[3,4].Theenvironmentalimpactof cigarettebutt wasteisrelatedtoboththeirpersistenceintheenvironmentand potentialtoxiceffects[5]andtheirchemicalcomposition− con-tainingover4000differentchemicals[4].Mostofthesechemicals aretoxicandoftenleachintoaquaticecosystems,thereby threaten-ingwatersupplysourcesandaquaticanimals[6,7]suchaswater fleas[3],which inhabitfreshwaterenvironments,andmosquito larvae[8]

Electroniccigarettes(EC)have,sincetheirmarketintroduction

in2004,gainedawideaudience−asof2013,thereareseveral mil-lione-cigaretteusersgloballyMichaelFelberbaum(11June2013) VariousreasonsorfactorshavecontributedtoincreasedECuse; includingtheperceptionthatvapingisasaferorhealthier alterna-tivetosmokingconventional(tobacco)cigarettes.Buttheimpact http://dx.doi.org/10.1016/j.toxrep.2017.01.003

2214-7500/© 2017 Published by Elsevier Ireland Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Plate 1.Control embryo top left Top right panel is RCBL (2CB/L) and the embryo exhibits minor malformations of head, gut and overall embryonic delayed development The RCBL embryo also exhibited significant tail abnormalities Bottom left is MCBL (0.5 CB/L) and the embryo exhibits severe edema in the heart, craniol-facial, and abdomial areas There are also significant gut and facial abnormalities Bottom Right panel is ECBL at 8 CB/L and the embryo exhibits some tail abnormalities The embryo has moderate

to severe edema, heart and gut abnormalities The embryo also has facial abnormalities as well.

ofECuseontheenvironmentisunknown.ECdeliveraerosolby

heatingaliquidsolutionknownase-liquidthatusuallycontains

nicotine,propyleneglycol,glycerin,andflavorings.TheECcombine

thefluidchamberwithaheatingelement,vaporizingthee-liquid

SinceECdonotreleasesecondhandsmoke,theyaretoutedbysome

asbeingasafealternative,forusersandbystanders,totraditional

(analog)cigarette.Butresearchershave analyzedtheimpurities

inbothe-liquid[9,10,11,12],andthecompositionoftheemitted

vapor[13,14,15,16].Goniewiczetal.[13]measured0.03–0.57␮gof

leadpere-cigaretteinemittedvapor.Williamsetal.[16]found

met-als(e.g.,lead,nickel,andsilver),silicatebeads,andnanoparticlesin

e-cigaretteaerosol.Thereisfarlessdataregardingthe

environmen-talimpactofECdisposal.WhileoneECistypicallyequivalentto40

traditionalcigarettes,greatlyreducingrelatedwastebyproducts,

someresearchhasbeenconductedtodeterminewhetherornotEC

wasteshouldbeclassifiedashazardous[17].KrauseandTownsend

[17]foundthatsomee-cigarettesmaybeclassifiedashazardous

wasteforleadleaching,thoughtheydidobservea largedegree

ofvariabilityamongbrandsandproducts.Theydidstressthatthe

rateofconsumptionanddisposaloftheseproductsisassumedto

behigherthanmanyotherelectronicproducts

Inordertoderivewaterqualityguidelinesorconducthazard

assessments,toxicitydataforanumberofaquaticspeciesisthe

minimumneeded,soitisimportanttodeterminenotonlythe

tox-icityoftraditionalcigarettebuttleachatebutalsothetoxicityof

electroniccigaretterelatedwaste.Inthisstudy,weexaminedacute

toxicity,teratogenicdevelopment,chronicandsublethaleffectsof

cigarettebuttleachateand ECleachateusingFrogEmbryo

Ter-atogenesis Assay− Xenopus(FETAX) FETAX is an inexpensive

alternativetoxicitytestsystemthatcanbeusedtoevaluate

ecolog-icalhazardsfrombothcomplexenvironmentalmixturesandpure

chemicalproducts[18].Thisbioassayisafour-day,whole

embryo-larvaldevelopmentaltoxicityscreeningassaythatusedembryosof

theSouthAfricanclawedfrog,Xenopuslaevis.TheFETAXsystemis

capableofmonitoringacute,chronic,developmental,and

behav-ioraltoxicityforecologicalandhumanhealthhazardassessment

Withover300validationtestcompounds,thepredictiveaccuracy

oftheFETAXmodelwithconventionalmammaliantestsystemsis

approximately85%[19].Thisallowsfortheexaminationofnotonly

acutetoxicity,butteratogenicdevelopmentandchronic,sublethal

effectsongrowth

Thespecificaimofthisstudywastodeterminehowdifferent

electroniccigarettebuttleachate(ECBL)werecomparedto

tradi-tionalcigarettebuttleachateintermsofenvironmental(aquatic)

toxicity,teratogenicityandimpactongrowthtoembryosof

Xeno-puslaevis.WehypothesizedthattheECBLwouldbelesstoxicthan thetraditionalcigarettebuttleachate

Theobjectivesofthisstudyweretodeterminetheacutetocity (96-hLC50and96-h EC50)and lowestobservedeffect concen-tration(includingtheminimumconcentrationtoinhibitgrowth)

ofregularcigarettebutt(RCBL),menthol(MCBL),andelectronic cigarettebuttleachate(ECBL)toXenopuslaevisembryos

l-cysteine, human chorionic gonadotropin (HCG), NaCl, NaHCO3, KCl, CaCl2, CaSO4, and MgSO4 and other laboratory supplies wereobtainedfromtheSigma ChemicalCo., St.Louis,

MO.Othersuppliesincluding60×15plastic Petridishes,10mL disposablepipettes,transferablepipettes,anda1mLsyringewere obtainedfromFisherScientificSupplies,location.Xenopuslaevis frogswereobtainedfromXenopusI,Inc.TheRegularandmenthol cigarettescontained2.8%and2.7%nicotinerespectively,andthe electroniccigarettecartridgescontained2.4%nicotine

1.1 Cigarettebuttcollection,electroniccigarettepurchase,and leachatepreparation

Cigarette butts (CB) were collected from naturally smoked cigarettes,definedascigarettesthatweresmokedbypeople, extin-guishedincigarettedisposalunitsandcollectedwithin24h.The electroniccigaretteswerepurchased,punctured,andthenused dis-cardedcigarettebutts.Weonlytestedonetypeofe-cigarette,as wellasonebrandofregularandmentholcigarette.TheCBwere selectedrandomlyandplacedintocleanplasticbagsfortransport

tothelaboratory.ThecollectedCBwereimmediatelyprocessedas describedbelowforleachateproduction

Cigarettebuttleachates(CBL)weremadebysoaking3buttsin

300mLFETAXsolutionfor1htoproducetheleachatestock solu-tionof10CB/L FETAXsolutionwaspreparedbyadding625mg NaCl,96mgNaHCO3,30mgKCl,15mgCaCl260mgCaSO4*2H2O, and75mgMgSO4perliterdistilledwater[19,20].CBLsweremade freshforRCBL,MCBL,ECBLforeachreplicateexperiment

1.2 Animalcareandbreeding Animalcareandbreedingwasperformedaccordingtothe spec-ificationssetforthintheASTM’sStandardGuideforConducting theFrogEmbryoTeratogenesisAssay-Xenopus[19].Animalcare andusewasperformedinaccordancewiththerequirementsof

Ciga rett e butt s / Liter

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mortality Malformation

Cigarett e butts / Liter

0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05

Fig 1. Representative concentration response graphs for Mortality & Malformation (Right Panel) and Growth (Left Panel) from a single experiment with Regular Cigarette Butt Leachate (RCBL) The Mortality & Malformation indicated an increasing response with concentration of RCBL The growth curve indicated decreasing length with increasing concentration of RCBL Error bars represent standard error.

Table 1

Individual analysis of each replicate experiment with 96-h LC50, EC50(malformation), Teratogenic index (TI), Minimum Concentration to Inhibit Growth (MCIG), Mortality and Malformation LOEC (Lowest Observable Effects Concentration) and NOEC (No Observable Effects Concentration).

CBs = cigarette butts Teratogenic Index (TI) = 96-h LC50/96-h EC50 Numbers in Parenthesis are 95% Fieller Bounds RCBL = Regular Cigarette Butt Leachate, MCBL = Menthol Cigarette Butt Leachate, ECBL = Electronic Cigarette Butt Leachate.

a All first experiments were conducted with the same clutch and all of the second experiments were conducted with the same clutch N.C.= Not Calculable.

theJacksonvilleStateUniversity’sInstitutionofAnimalCareand

UseCommittee.Adultfrogswerekeptinglassaquariumsin

re-circulatedwateratroomtemperature(24±2◦C).A12-hday/12-h

nightcyclewasmaintained.Malesandfemaleswerebredas

sin-glepairs.Breedingwasinducedbyinjectingeachfrogwithhuman

chorionicgonadotropinandplacedinfalsebottombreeding

cham-bers[21].Malesreceived250–500IUofHCG,andfemalesreceived

500–1000IU.Thehormoneconcentrationwas1000IU/mLinsterile

0.9%NaClandinjectedwitha1-mLtuberculinsyringefittedwith

a½inchlong26-gaugeneedle[19]

FETAX procedures were performed according to ASTM’s

Standard Guide for Conducting the Frog Embryo

Teratogene-sis Assay-Xenopus [19] Eggs were collected and immediately

inspected todeterminefertility and quality Thejelly coatwas

removedbyswirlingtheeggsfor 1–3minin 2%w/vl-cysteine

solutionpreparedinFETAXsolution,andpHwasadjustedto8.1

with1NNaOH.Immediatelyafterthejellycoatwasremoved,l

-cysteine wasrinsedoff withFETAXsolution.Healthy,normally

dividingblastulaewereplacedin60×15plasticPetridishes,each

dishcontaining20embryos.Foreachtest,stocksolutionsofCBL

weremadefresh daily.Dead embryoswereremoveddaily and

alivecountwastaken.Attheend ofa four-dayperiod,a final

live/deadcountwastakenandthenumberandtypeofmalformed

wasrecorded.EmbryoswereanesthetizedwithMS222and,then

fixedin3.0%(w/v)formalin.Malformationtypeswerenotedon

theScoresheetofMalformationsat96-hasdescribedintheFETAX

AtlasofAbnormalities[22].Embryoswerephotographedand

mea-sured forlength usingImage Pro plus software.Two definitive

dose-responsetestswereperformed.Thesameclutchwasusedfor

eachofthedefinitive1andasecondclutchwasusedfordefinitive

2forallleachatetypes

1.3 Statisticalanalysis Meansandstandarderrorwerecalculated.The96-hLC50,96-h EC50forindividualandcombinedexperimentsweredetermined usingprobitanalysisbySystat13.TheTeratogenicIndex(TI)was calculatedbydividingthe96-hLC50/96-hEC50.Embryolength, malformation,andmortalitydatawereanalyzedwithANOVA fol-lowedbyBonferroni’st-testformultiplecomparisonstodetermine theMinimumConcentrationtoInhibitGrowth(MCIG),TheNoand LowestObservableEffectsConcentration(NOEC,LOEC)for mortal-ityandmalformation

Theoverallaveragecontrollengthsrangedfrom0.96to1.03cm Thetypicalmalformationsseeninthecontrolsincludedabnormal gutcoiling,slightedema,andsometailandnotochord malforma-tions.Fortheoverallexperiment,56ofthe480controltadpoles exposeddied,yieldinga11.6%mortalityrate.Ofthe424surviving tadpoles,17weremalformedforanoverallmalformationrateof 4.0%

2.1 Regularcigarettebuttleachate(RCBL) The Mortality and Malformation showed a sharp increase concentrationresponse withMortalityandMalformationcurves

Cigarette butts / Liter

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Mortality Malformation

Cigarette butts / Liter

0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05

Fig 2.Representative concentration response graphs for Mortality & Malformation (Right Panel) and Growth (Left Panel) from a single experiment with Menthol Cigarette Butt Leachate (MCBL) The Mortality & Malformation indicated an increasing response with concentration of MCBL The growth curve indicated decreasing length with increasing concentration of MCBL Error bars represent standard error.

followingeach other.(Fig.1 LeftPanel).The96-h LC50forthe

twoclutchesofembryosexposedtoRCBLwas0.68–1.65CBs/L.The

96-hEC50forthetwoclutchesofembryosexposedtoRCBLwas

0.34–1.21CBs/L.TheTeratogenicIndex(TI)forthetwoclutcheswas

1.36–1.95CBs/L.TheMinimumConcentration toInhibitGrowth

(MCIG)forRCBLwascalculatedtobe0.25and0.5CBs/L(Table1)

Therewasa sharp direct negativecorrelation betweenembryo

lengthand regular cigarette buttleachate concentration(Fig 1

RightPanel).Thereductioninlengthofcontrolscomparedtothe

highestsurvivingconcentrationwas0.26cm.TheLowestandNo

ObservableEffectsConcentrations(LOEC,NOEC)forMortalitywere

consistentwith 0.5 CBs/L for the LOEC and 0.25 CBs/L for the

NOEC.ForMalformation,theNOECwasmorevariableduetoclutch

differences.DominantmalformationsforRCBwereloosegut

coil-ing,facialmalformations,notochordmalformations,andstunting

(PhotoPlate1,TopRightPanel)

2.2 MentholCigaretteButtLeachate(MCBL)

TheMortalityandMalformationshowedanincrease

concen-trationresponsewithadistinctseparation ofthemortalityand

malformation curves (Fig.2 left panel) The 96-h LC50 for the

twoclutches of embryoswasconsistentat just over1.078 and

1.140CBs/Lindicatinggoodagreementbetweenthetworeplicated

experiments.Butthe96-hEC50showedalittlemorevariability

between0.3–0.96CBs/L.Itisthedifferencesinthe96-hEC50that

accountfortheTIbeingfrom1.43to3.77.The MCIGwas

con-sistentat0.5CBs/L.Therewasa sharpdramaticdirectnegative

correlationbetweenembryolengthandMCBL(Fig.2rightpanel)

Thereductioninlengthofcontrolscomparedtothehighest

surviv-ingconcentrationwas0.21cm.Thegrowthreductionwas0.20cm

forthehighestsurvivingconcentrations.Themalformation

end-pointwasthe mostsensitivewiththeLOECbeing0.25 to<0.1

CBs/L.Themalformations seenwithMCBLweremostdramatic Therewassignificantconsistentedema,gut,heart,andcraniofacial abnormalities(PhotoPlate1bottomleftpanel)

2.3 ElectronicCigaretteButtLeachate(ECBL) TheMortalityandMalformationshowedauniqueconcentration responsewithmortalityandmalformationcurvesoverlappingfor mostoftheconcentration(Fig.3leftpanel).Therewasonepoint

at0.5CBs/Lthatshowedadramaticdropinembryolength; how-ever,itwasnotconsistentwithotherconcentrationstested.The 96-hLC50indicatedalowtoxicitywith9.7and26.8CBs/L.The twoexperimentsweredifferentwiththesecondshowingmuch lesttoxicitythanthefirst.Neitherexperimentyieldedmany mal-formationsatallconcentrationstested.TheTIoftheexperiment indicatedwidelydifferentnumbersof0.47–1.72duetothechange

inthe96-hLC50ofExperiment2.TheMCIGwasrightatthe high-estconcentrationtestedof10CBs/Lorhigher.Embryogrowthwas negativelyimpacted;however,thegrowthcurvehadamuchmore gradualslope(Fig.3rightpanel).Thereductioninlengthofcontrols comparedtothehighestsurvivingconcentrationwasonly0.05cm ThemalformationsseenwithECBLwereslightedema,loosegut coiling,andsometailabnormalities(PhotoPlate 1bottomright panel).Thesemalformationsweremoderatetoseverewhenthey occurred

Thisresearchindicatestheoverall96-hLC50ofRCBLandMCBL was1.2and1.14CBs/Lrespectively(Table2).Theoverall96-hEC50 (malformation)ofRCBLandMCBLwas0.90and0.50CBs/L(Table2)

Itwouldbeexpectedthatembryoswithmalformationswouldnot surviveintheenvironment.Thesevaluesarewithintherangeof

Table 2

Overall summary analysis of Regular Cigarette Butt Leachate (RCBL), Menthol Cigarette Butt Leachate (MCBL), and Electronic Cigarette Butt Leachate (ECBL) with the 96-h LC50, EC50 (malformation), Teratogenic index (TI), Minimum Concentration to Inhibit Growth (MCIG), Mortality and Malformation LOEC (Lowest Observable Effects Concentration) and NOEC (No Observable Effects Concentration).

Ciga rett e butt s / Liter

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Mortality Malformation

Cigarett e butts / Liter

0.86 0.88 0.90 0.92 0.94 0.96 0.98 1.00 1.02 1.04

Table 3

Ceriodaphnia cf dubia 0.06–1.7 CBs/L LC 50 [23]

Ceriodaphnia cf dubia 0.03–0.08 CBs/L EC50 [24]

Vibrio fisheri 0.58–1.25 CBs/L LC50 [23]

Vibrio fisheri 0.3–2.7 CBs/L EC50 [24]

previouslytestedaquaticspecies(Table3).Thisdemonstratingthat

Xenopuslaevisembryosaregoodindicatorsoftoxicityassociated

withcigarettebuttleachate.Inaddition,theFETAXassayshowed

muchlowerCBs/LforotherendpointsmeasuredsuchasMCIGof

0.01and0.25CBs/L(Table2)

3.1 ImpactofRCBLandMCBL

TheRCBLandMCBLhadanoverall TIof 1.33–2.28(Table2)

indicatingthatthereissometeratogenicriskassociatedwiththe

leachatewith MCBLhaving morerisk This would beexpected

becausenicotineisknowntobehighlyteratogenicintheFETAX

assay[25].ThisindicatesthattheCBLareapotentialteratogenic

hazardtoaquaticlifeinrivers,streams,andlakesand[agreeswith]

isconsistentwithdatafromLeeandLee[26].TheMCBLindicates

ahigherriskduetoeitherthementholpresentorpossibly

inter-actionsoftheadditiveswiththenicotineandotherproductsfrom

smokingcigarettes.Anotherpossibilityisthattheadditives

some-howaddedtheabilityofnicotineandotherproductstoleavethe

cigarettes.ThisdoesnotsupportthestatementbySlaughteretal

[4]that“Thereisnoresearchtosupportthatflavoredcigarettes(eg,

menthol)altertoxicityorimpartadditionaltoxicity.”Althoughthe

overalltoxicitydifferencesarenotlargetheimpacton

malforma-tionswassignificant

TheECBL wasmuch lesstoxic withanoverall 96-h LC50of

14.6CBs/L(Table2)indicatingthattheECBLwasatleast10fold

lesstoxicthantheregularcigarettebutts.Surprisingly,the96-h

EC50(malformation)wasevenhigherat16.3CBs/L.Theoverall

ter-atogenicindexwas0.90indicatingtherewasalsolittleteratogenic

risk.BecausetheECBLwouldbeexpectedtocontainnicotine,this

mightmeanthatECBsmightnotallowthenicotinetobe

bioavail-ableorsomeothermechanismsareinplacetopreventexcessive

nicotineexposuretotheenvironment.Therewasmuchlesseffect

ongrowthasindicatedbytheveryhighMCIGcomparedtothe

traditionalcigarettebutts

Electroniccigarettesareapproximately10foldlesstoxic Xeno-pus embryosthan traditional cigarettes Thisindicates that the impactofdiscardedECBonaquaticenvironmentswould poten-tiallybelessthanthatoftraditionalcigarettebutts.Theimpactis furtherminimizedbythefactthat1ECBisequivalentto approx-imately40traditionalcigarettes[17].Theaquaticenvironmental impactofECBsthuscouldbepotentially400timeslessthanthat

oftraditionalcigarettes.Themethodsthatwereusedinthisstudy, however,wereonlya1hsoaking/mixingtimewithECBthathad notbeenphysicallydamagedastheymightbeinalandfill.This datawouldalsonotincludepotentialimpactsbylong-termmetal erosionorbatterdegradationintotheaquaticenvironmentamong otherfactors.Thepurposeofthispaperwastoexaminewhatwould leakoutofeasilydiscardedcigarettebutts,andnotonmore elab-orateelectroniccigaretteswithrefillablecartridges.Thepathways

ofthiscigarettewastetoaquaticenvironmentsarecomplexand varied,andthatmakescorrelationstoriskdifficulttostudyand determine[4]

FETAXproved tobea useful testing assayfor cigarettebutt leachateandisviableforfurthertesting.Becausetraditional men-tholcigarettesresultedindifferentresultsthanregularcigarettes, especially with regards to gross malformations, future studies shouldexaminementholatedcigarettesinmoredetail.Inaddition, longersoaking/mixing times,as wellas testing the liquid con-tentsofrefillablee-cigarettes(flavoredandnon-flavored),should

beassessed

Acknowledgement

Wewould liketothankJacksonvilleStateUniversityBiology Departmentforsupportofthisproject

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