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Developing earthworm bioconcentration factors of nitrogen-based
compounds for predicting environmentally significant parameters for
new munition compounds in soil
Kuperman, Roman G.; Simini, Mike; Checkai, Ronald T.; Dodard, Sabine G.;
Sarrazin, Manon; Al-Hawari, Jalal; Paquet, Louise; Sunahara, Geoffrey I.; Di
Toro, Dominic M.; Allen, Herbert E.; Kuo, Dave Ta Fu; Torralba-Sanchez,
Tifany L.
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Developing
earthworm
bioconcentration
factors
of
nitrogen-based
compounds
for
predicting
environmentally
significant
parameters
for
new
munition
compounds
in
soil
Roman
G.
Kuperman
a,*
,
Mike
Simini
a,
Ronald
T.
Checkai
a,
Sabine
G.
Dodard
b,
Manon
Sarrazin
b,
Jalal
Hawari
b,c,
Louise
Paquet
b,
Geoffrey
I.
Sunahara
b,d,
Dominic
M.
Di
Toro
e,
Herbert
E.
Allen
e,
Dave
Ta
Fu
Kuo
f,
Tifany
L.
Torralba-Sanchez
eaU.S.ArmyEdgewoodChemicalBiologicalCenter,AberdeenProvingGround,MD21010-5424,UnitedStates bNationalResearchCouncilofCanada,Montreal,QCH4P2R2,Canada
cDepartmentofCivil,GeologicalandMiningEngineering,PolytechniqueMontreal,Montreal,QCH3C3A7,Canada dDepartmentofNaturalResourceSciences,McGillUniversity,Sainte-Anne-de-Bellevue,QCH9X3V9,Canada eDepartmentofCivilandEnvironmentalEngineering,UniversityofDelaware,Newark,DE19716,UnitedStates fDepartmentofArchitectureandCivilEngineering,CityUniversityofHongKong,HongKong,China
ARTICLE INFO Articlehistory:
Received30September2014
Receivedinrevisedform12March2015 Accepted13November2015 Availableonlinexxx Keywords: Munitioncompounds Bioconcentration Toxicity Earthworms ABSTRACT
Weinvestigatedthebioconcentrationpotentialofnitrogen-basedcompounds4-nitroanisole(4-NAN), 3,5-dinitro-o-toluamide(3,5-DNoTAME),and2-methoxy-5nitropyridine(2-M-5-NPYNE)using
earth-wormEiseniaandreiexposuresin aqueousexposuremediainsand. Separatetoxicitystudieswere
conductedpriortobioconcentrationstudiesusingarangeofchemicalconcentrationstoestablishthe sublethalexposureconditionsfortheearthworms.Theobjectivesofthepresentstudieswereto:(1) developanexperimentaltestsystemforestimatingbioconcentrationpotentialsofnewandemerging munitioncompoundsthatpartitionintoearthworms,usingaqueousexposuremedia;and(2)applythis experimentalmodeltoestablishoriginalbioconcentrationdatafor4-NAN,3,5-DNoTAME,and 2-M-5-NPYNE.Experimentaldesignincludesearthwormexposurestochemicalsforupto14daysinaqueous media(Römbkemedium;0.08mMKCl,2mMCaCl2,0.5mMMgSO4,and0.8mMNaHCO3)inthepresence
ofwater-washedcoarsesand(0.5–1.0mm)substrate.Concentrationsoftestchemicalsinrespective exposuremediaremainedrelativelystableduringtheseindependentstudies.Tissueanalysesrevealeda rapid uptake of eachchemical by theearthworms; asteadystate was attainedwithin 24h from commencementoftheseexposures.Estimatedsteady-statebioconcentrationfactors(BCFss;mL/gdry
tissue)were47,6,and11for4-NAN,3,5-DNoTAME,and2-M-5-NPYNE,respectively.Theseresultswill contributetotheBCFdatabasebeingdevelopedforuseinmodelsaimedatpredictingenvironmentally significantparametersfornewmunitioncompoundsinsoil.
PublishedbyElsevierB.V.
1.Introduction
Anewgenerationofhighlyenergeticchemicalsisexpectedto replacetraditionalexplosivesandpropellants.Examplesinclude newshock-insensitivemunitions(IM)compoundsthatminimize theprobabilityofinadvertentdetonationasaresultofaccident, combat,orterrorist actions.Toaddress apressing needfor the United States Department of Defense (DoD) to reduce the environmental risks associated with military operations,
investigationsofenvironmentalfateandecotoxicologicaleffects ofsomeofthesecompounds,suchas2,4-dinitroanisole(DNAN) andnitro-triazolone(NTO),havebeeninitiated(Ampleman,2010; Amplemanetal.,2012;DaviesandProvatas,2006;Dodardetal., 2013;Haleyetal.,2009).However,ecotoxicologicaldatadeveloped inthesestudiesarepertinentonlytotheexistingcompounds.A more critical need in reducing the environmental risks of energetics is the development of new approaches to predict environmental fate,suchas uptakebysoil organisms,of future compoundswhiletheyarestillintheinitialdesignphases.
Theuseofquantitativestructureactivityrelationships(QSARs) to estimate the environmental and toxicological properties of potential contaminants hasbeenan activearea ofresearch for
* Correspondingauthor.
E-mailaddress:roman.g.kuperman.civ@mail.mil(R.G.Kuperman).
http://dx.doi.org/10.1016/j.apsoil.2015.11.008
0929-1393/ PublishedbyElsevierB.V.
Pleasecitethisarticleinpressas:R.G.Kuperman,etal.,Developingearthwormbioconcentrationfactorsofnitrogen-basedcompoundsfor ContentslistsavailableatScienceDirect
Applied
Soil
Ecology
manyyears(VanGestelandMa,1990;Selassie,2003).However, predicting bioconcentration factors (BCFs) of new munition compoundsischallengingbecausemodelscurrentlyavailabledo notaccuratelyaccountforeitherpartitioningofacompoundinto anorganismordegradation.Furthermore,predictingtheBCFfrom soilexposurestosoilbiotaisdifficultbecausesoilpropertiesaffect thebioavailabilityofthecompound(Dodardetal.,2005; Kuper-manetal.,2009,2013;Lannoetal.,2004;Rocheleauetal.,2010; Sunaharaetal.,2009).Thisparticularchallengecanberesolved,in part,bytheuseof aqueousexposures,thusseparating thesoil effectsfromtheorganismuptakemechanismswhendeveloping empiricaldata for use in models.Therefore, we conductedthe present experiments to develop the necessary uptake data for selectedcompoundsforuseinmodeldevelopment.Theselected compounds that have nitro-group functionality can serve as proxies for new munitions. These compounds share strong electron NO2 withdrawing groups, which are important in
determining the environmental fate, transport, transformation, and ecological effects of the explosives (Monteil-Rivera et al., 2009).Compound-specificpropertiesaresummarizedinTable1. Totest thehypothesisrelatingtheuptake ofselected nitrogen-based organic compounds to their molecular structures, we designedthepresentstudiesto:(1)developanexperimentaltest system for estimating bioconcentration potentials of new and emergingmunition compounds thatpartition intoearthworms, usingaqueousexposuremedia;and(2)applythisexperimental modeltoestablishoriginalbioconcentrationdatafor4-NAN, 3,5-DNoTAME,and2-M-5-NPYNE.
2.Materialandmethods
2.1.Testspecies
Theearthworm(Oligochaeta,Annelida)EiseniaandreiBouché (1972)wasusedinthepresentstudies.Earthwormswerebredin plasticcontainersfilledwithapproximately14kgofa1:1mixture ofPRO-GROsphagnumpeatmoss(GulfIslandPeatMossCo.,PEI, Canada)andBACCTO1pottingsoil(MichiganPeatCo.,Houston,
TX, USA). The pH was adjusted to 6.20.1 by adding calcium carbonate (pulverized lime). The culture was kept moist at 212C, undercontinuous light.Earthwormcolonies werefed
biweeklywithalfalfafood,consistingofdehydratedalfalfapellets (27%fiber,17%protein,1.5%fat;OhioBlendersofPA,York,PA)that werepreparedbyhydrating,fermentingforatleast14days, air-dried,andthengroundtoacoursepowder.Earthwormcultures were synchronized so that all worms used in each test were approximately thesame age. Individual earthworms withfully developedclitella, and weighingfrom0.3 to0.6g(wet weight)
wereallowedtodepuratetheirgutcontentonmoistfilterpaperfor atleast24h,andthenrandomlyassignedtoeachtestcontainer. Measurement endpoints in toxicity tests included adult earth-wormsurvivalandwetweight.
2.2.Exposuremedia
Preliminarystudieswereconductedtoselecttheappropriate exposure mediumfor subsequenttoxicityand bioconcentration studies.Formulationsofaqueousexposuremediawereprepared accordingtoRömbkeandKnacker(1989;theRömbkemedium)or
Robidouxetal.(2002;theLumbricusBalancedSaltSolution(LBSS) medium). The Römbke medium contained 0.08mM KCl, 2mM CaCl2,0.5mMMgSO4,and 0.8mMNaHCO3.TheLBSScontained
71.5mMNaCl,4.8mMKCl,3.8mMCaCl2,1.1mMMgSO4,0.4mM
KH2PO4,0.3mMK2HPO4,and4.2mMNaHCO3.Solutionsofeach
medium were prepared using ASTM type I water (ASTM International,2004).TheControlmediumconsistedofASTMtype I water only. Each aqueous exposure medium was replaced regularly(1–3dayintervals)duringthestudies.Solidcomponents wereincludedinthestudiestodeterminewhetheravailabilityof solid substrate saturated with the aqueous exposure medium extendsthe survival of theearthworms. The solid components tested were either coarse sand (0.5–1.0mm), or glass beads (10mm).Sandandaqueousmediumwerechangeddaily,whilethe glass beads were rinsed with ASTM type I water or Römbke medium,respectively,every1–3days.Thesepreliminarystudies showedthatearthwormsurvivalwasgreatestinasand-Römbke exposuremediumfor 28d (datanotshown).Consequently,this mediumwasselectedfortheearthwormtoxicityand bioconcen-trationstudieswithtestchemicals.
2.3.Toxicitytests
Noecotoxicologicaldatawereavailableforthetestcompounds; therefore, toxicity tests were conducted to determine the appropriate exposure concentration of each test compound for useinthebioconcentrationstudies.Aconcentrationwasdeemed appropriatewhenitsustainedsurvivaloftheearthwormsforthe durationofthetest.Separatetoxicitytestswereconductedwith 4-nitroanisole (4-NAN; an intermediate in the manufacture of syntheticorganicdyesandpharmaceuticals), 3,5-dinitro-o-tolua-mide(3,5-DNoTAME;usedasaveterinarycoccidiostat(tocontrol coccidiosis)andfoodadditive),and2-methoxy-5nitropyridine (2-M-5-NPYNE;usedasaprecursorinorganicchemistrysyntheses). Toxicitytestsincludedexposingearthwormstoarangeofchemical concentrations in sand-Römbke medium for up to 21 days. Nominal/analyticallydeterminedtreatmentconcentrations(mg/
Table1
Selectedpropertiesoftestcompounds4-nitroanisole(4-NAN),3,5-dinitro-o-toluamide(3,5-DNoTAME),and2-methoxy-5nitropyridine(2-M-5-NPYNE).
Name 4-NAN 3,5-DNoTAME 2-M-5-NPYNE
CAS-Reg.Number 100-17-4 148-01-6 5446-92-4
Molecularmass(g/mol) 153.1 225.2 154.1
Structuralformula
LogKow(L/kg) 2.03 0.19 1.55
Watersolubilitya
(mg/L)
552.9 447.5 1406
aValuesbasedontheU.S.EPAEstimationProgramsInterfaceSuite(EPISuite);estimatedat25C.
2 R.G.Kupermanetal./AppliedSoilEcologyxxx(2015)xxx–xxx
L)were6/11,29/65,58/131,116/223,and232/289for4-NAN;5/10, 48/100,96/141,and 192/178for3,5-DNoTAME,and6/11,56/117, 113/178, 227/216 for 2-M-5-NPYNE. The experimental control treatmentconsistedofRömbkemediumwithnotestchemicals added.Alltreatmentswerereplicated(n=3).Thetoxicitytestwith 2-M-5-NPYNE was repeated using nominal treatment concen-trations of 2.3, 4.7, and 9.4mg/L due to 100% mortality at all nominalconcentrations56mg/Lintheinitialtest.Corresponding analyticallydeterminedconcentrationsof2-M-5-NPYNEwere14, 25,and50mg/L,respectively.
2.4.Bioconcentrationstudies
The design of the bioconcentration studies followed the protocol established in the international ring test for the assessmentofbioaccumulationofchemicalsinterrestrial oligo-chaetes(Brunsetal.,2001;Egeleretal.,2009).Thismethodwas modifiedforaqueousexposuresoftheearthworms.TheBCFswere determinedusingastaticaqueousexposuretestsystemsimilarto theapproachbyBelfroidetal.(1993).Thebioconcentrationstudies includedexposuresoftheearthwormstoindividualtestchemicals inRömbkemediuminthepresenceofsandforupto14days.This experimental design was applied to studies with 4-NAN, 3,5-DNoTAME,and 2-M-5-NPYNE.Thestudydesign alsoincludeda controlgroup(Römbkemediumwithnotest chemicalsadded), andalipidgroup(inbothcontrolandtestchemicaltreatments) usedtoquantifyearthwormlipidcontents.
Glass jars (4.2cm i.d.4.5cm height) were used as test containers.Eachtestcontainerwasfilledwith7.4mLofexposure mediumand 21gof water-washedcoarsesilica sand.All treat-mentswereappropriatelyreplicated(n=5perharvestday).Two depuratedearthworms wereexposed in each replicate.Alltest containers were placed in an environmental chamber under a meantemperatureof21.60.1Cinthedarkforthedurationof
the14daytest.Theexposuremediainalltest containerswere renewedevery2-3days.Replicatesofeachtestchemicaltreatment wereharvestedafter0,0.25,1,2,3,4,7,and14daysofexposure. Theconcentrationsoftestcompoundintheaqueousmediawere analyticallydeterminedforeachharvest.Earthwormswererinsed withASTMtypeIwaterandpaper-driedtoremoveexcesswater. Wetweights of theearthwormswere recorded,and the earth-worms were then frozen at 80C. Frozen earthworms were
lyophilizedfor7dayspriortoresidueanalysisandlipidcontent determinations. Uptakeof test compounds by the earthworms fromsolutions was quantified understeady-state conditions to determinetheempiricalBCFssvalues(theratioofconcentrationsin
tissuecompared toexposure media([C]tissue/[C]media).A
steady-stateconditionwasacceptedwhentestchemicalconcentrationsin theearthwormtissuesstabilized.Stabilitywasreachedwhenthere werenosignificant(p>0.05)differencesintissueconcentrations foratleastthreeharvestdatesattheendofthestudy.
2.5.Chemicalanalysesandanalyticaldeterminations
A modified U.S. Environmental Protection Agency (USEPA) Method 8330B(USEPA,2006)was usedto extractand quantify the concentrations of test chemicals in exposure media at predetermined time points. Tissue extracts of the earthworms respectivelyexposedto4-NAN,3,5-DNoTAME,and2-M-5-NPYNE werepreparedaccordingtoSarrazinetal.(2009).Earthwormsin thecontrolgroupswereexposedtothesand-liquidmediawithout chemicals.Foreachreplicate,allearthwormswerelyophilizedat theendofeachexposureinterval,thencrushedusingamortarand pestletoobtainfrom0.1to0.2gofdrymaterialforanalyses.The crusheddrytissuewasthenextractedusingamixtureofASTMtype Iwaterandacetonitrileat4C.Eachtissuesamplewasvortexedin
ASTM type I water for 10s before addition of acetonitrile. All sampleswerethenvortexedforanadditional60sandsonicated (BransonModel3200,Danbury,CT,USA)for182hat20C,then
centrifuged(12,000g)for 10minat4C usinga Sorval Super
T21centrifuge(GlobalMedicalInstrumentation,Ramsey,MN,USA).
Time (days) 0.00 0.05 1.00 2.00 3.00 4.00 7.00 4 -NA N ( m g /k g ) 0 10 20 30 40 Time (days) 0.00 0.05 1.00 2.00 3.00 4.00 7.00 14.00 3, 5-D N o TA M E ( m g /k g ) 0 2 4 6 8 10 12 14 16
Time (days)
0.00 0.05 1.00 2.00 3.00 4.00 7.00 14.00 2-M -5-N P Y N E ( m g /k g) 0 2 4 6 8 10 12
A
B
C
Fig.1.Concentrationsof4-NAN(A),3,5-DNoTAME(B),and2-M-5-NPYNE(C)in aqueousexposuremediaduringthebioconcentrationstudies.
TheHPLCsystemusedforanalysesconsistedofaWaters600pump (Waters Corp, Milford, MA), a 717 plus autosampler, and a 2996photodiode-ArrayDetector.Samples(50
m
L)wereseparatedwitha Discovery C18 column (25cm, 4.6mm, 5mm) (Supelco, Oakville,Canada)at35C.Thedetectorwassettoscanfrom192to
450nm.TheanalyticaldetectionlimitusingHPLCwas0.005mg/L foreachcompound. Thelimitsofquantificationwere0.6mg/kg tissuefor4-NAN,0.3mg/kgtissuefor3,5-DNoTAME,and0.3mg/kg tissuefor2-M-5-NPYNE.
2.6.Determinationoflipidcontentinearthworms
Depurated earthworms from the laboratory culture, and samples from the uptake studies were frozen at 80C
(7days), andlaterthawed onice forlipidextraction.One or two earthworms were weighed directly in a FalconTM
50mL polypropyleneconicalcentrifugetube.EightmLofisopropanol were added to the tube and vortexed for 5s then 10mL of cyclohexanewasaddedandthecontentinthetubewasvortexed for an additional 5s. Earthworms in cyclohexane/propanol mixturewerehomogenizedusingthehighspeedhomogenizer UltraTurraxT25for2minat10,000g.Avolumeof10.5mLof ASTMtype I water was added to the extraction mixtureand homogenizedforanother1min.FivemLofconcentratedHClwas addedtodissolvethemilkysuspensionpriortocentrifugationat 460gfor5min.Twophaseswereformed;thetoporganiclayer (transparentandyellowish)was transferredintoa pre-weighed vial. The bottom layer was cleaned by addition of 10mL of a cyclohexane/isopropanol (87/13; v/v) mixture, vortexed for 5s, then centrifuged at 460g for 5min. The resulting top layer (transparentand yellowish)wastransferred intothesame pre-weighedvialcontainingtheprevioustoplayer.Thiscleaningstep was repeated twice (normally) to remove traces of lipid, as evidencedbythetoplayerbecomingmoretransparentaftereach cleaningstep.Theextractwasthenevaporatedtodrynesswiththe nitrogenstream.Allvialswereplacedintoanovenat105Cfor1h.
Thevialswerecooledinadesiccatorandweightswererecorded, thentheywereplacedintoanoventodryagainforanother2h. Thecontents of each vial weretransferred into a pre-weighed aluminumcup, using<5mLof cyclohexane. Thesample inthe aluminum cup was concentrated to dryness under nitrogen stream, and placed into an oven at 105C, overnight. After a
maximumof16h, thedriedaluminum cups andtheircontents werecooledinadesiccator,andtheweightswererecorded.Lipid weightswerecalculatedbydifference.
2.7.Dataanalyses
Analysis of variance procedures were applied to the adult earthwormsurvival(exceptwhen 0or100%survival occurred), weight,lipidcontent,andtestchemicalconcentrationdata.Mean separationsweredoneusingFisher’sleast-significantdifference (FLSD)pairwisecomparisontests.Asignificancelevelofp0.05 (95% confidence level) was accepted for all statistical tests. Statistical analyses were performed using SYSTAT 11.0 (Systat Software,Inc.,Chicago,IL)orSigmaPlot10.0(SystatSoftware,Inc., SanJose,CA).
Resultsfromtheearthwormuptakestudieswerefittedusing theequationforaone-compartmentmodel:
C
½ wormattðiÞ¼½Cssf1 eðktÞg;
where[C]wormis thetest compoundconcentrationmeasuredin
earthwormtissues(mgchemical/kgdrytissue);tisuptaketimein d;[C]ssistheconcentrationofthecompoundinearthworm(mg
chemical/kgdrytissue)atsteadystate;kistheuptakecoefficient (1/day).
0
1
2
3
4
5
6
7
Time (da
ys)
0
500
1000
1500
4 -N A N i n tis s ue (mg/ k g dr y tis s u e )0
1
2
3
4
5
6
7
0
500
1000
1500
Fig.2. Uptakeof4-NANinearthwormtissueduring7dayexposureinstructured aqueousmedium.
0
2
4
6
8
10
12
14
Time (da
ys)
0
20
40
60
80
100
3 -5 -D N o T A ME i n ti s s ue ( m g/ k g dr y tis s u e )0
2
4
6
8
10
12
14
0
20
40
60
80
100
Fig.3.Uptakeof3,5-DNoTAMEinearthwormtissueduring14dayexposurein structuredaqueousmedium.
0
2
4
6
8
10
12
14
Time (da
ys)
0
26
52
78
104
130
2 -M -5 -N PY NE in tis s ue (mg/ k g dr y tis s ue)0
2
4
6
8
10
12
14
0
26
52
78
104
130
Fig.4.Uptakeof2-M-5-NPYNEinearthwormtissueduring14dayexposurein structuredaqueousmedium.
4 R.G.Kupermanetal./AppliedSoilEcologyxxx(2015)xxx–xxx
3.Resultsanddiscussion
3.1.Aqueoustoxicitystudies
Mean earthworm survival (SE) in the control and 29mg/L nominaltreatmentsfor4-NANwas1.30.67(n=3).Earthworm mass loss was 0.1 0.05g for the control treatment and 0.20.03 for the 29mg/L treatment. No earthworms survived exposuresto4-NANconcentration58mg/L.Inastudywith 3,5-DNoTAME, the mean survival rateswere 1.30.67 in both the controlandthe5mg/Ltreatments.Earthwormssurvivedinoneof thethreereplicatesusedinthe50mg/Ltreatmentconcentration. Themeanearthwormmasslosswas 0.1 0.05gfor thecontrol treatmentand 0.20.03g for the 5mg/L treatment. No earth-wormssurvivedineither96mg/Lor192mg/Ltreatment concen-trationsof3,5-DNoTAME.Allearthwormssurvivedinthetoxicity studywith2-M-5-NPYNE.Basedontheresultsofthesetoxicity tests, the following nominal exposure concentrations were selected for the bioconcentration studies: 30mg/L for 4-NAN, and 10mg/L for either 3,5-DNoTAME or 2-M-5-NPYNE. Corre-spondinganalyticallydeterminedexposureconcentrationsatthe startoftheindependentbioconcentrationstudieswere36mg/Lfor 4-NAN,15mg/Lfor3,5-DNoTAME,and11mg/Lfor2-M-5-NPYNE, respectively.
3.2.Bioconcentrationstudies
Chemicalanalysis revealedthat 4-NAN concentration in the exposure medium rapidly decreased (by 38%) from the initial exposureconcentrationof36mg/Lfollowingintroductionofthe earthworms,then stabilized after24h, and remainedrelatively constantfortheremainderofthe7-daystudy(Fig.1A). Concen-trationsof3,5-DNoTAMEintheexposuremediumalsodecreased after 6h from the initial exposure concentration of 15mg/L, following introduction of the earthworms, but the decrease continuedfor2d (31%fromtheinitial15mg/L). Concentrations of 3,5-DNoTAME stabilized after 3d and remained relatively constantfortheremainderofthe14-daystudy(Fig.1B).Arapid decrease(22%fromtheinitial11mg/L),duringthe6hperiodafter introductionoftheearthwormswasfoundinthestudywith 2-M-5-NPYNE. Similar to the pattern of results for 4-NAN, the concentrationsof2-M-5-NPYNEstabilizedafter24handremained relativelyconstantfortherestofthe14-daystudy(Fig.1C).
Tissueanalysesrevealed rapiduptake bytheearthwormsof each test chemical following earthworm introduction into the exposuremedium(Figs.2–4).Theseconcentrationscorresponded to rapid decreases in each test chemical concentration in the exposure medium. Steady-state conditions were attained in concentrationsinrespectiveearthwormtissuesforthefinalthree ormoreharvestdaysin theseindependentstudieswith4-NAN (p0.292), 3,5-DNoTAME(p0.160), and 2-M-5-NPYNE (p 0.110). Steady-state concentrations of each test chemical in theearthwormtissues wereused tocalculateBCFssvalues.The
estimatesoftheBCFssvalues(mL/gearthwormdrymass)for
4-NAN, 3,5-DNoTAME, and 2-M-5-NPYNE were 47, 6, and 11,
respectively. The correspondingestimates of the lipid based L-BCFssvalueswere332mL/g,58mL/g,and90mL/gearthwormlipid,
respectively(Table2).
The present bioconcentration studies have allowed us to separate the confoundingfactor of soil-waterpartitioning from that of organismexposure. Thiswas necessarybecause experi-mentalevidencesuggeststhatthedissolvedformofachemicalin interstitial or porewater maycontrol its bioaccumulation from soils or sediments. Several studies demonstrated that the bioavailabilityofmanyorganiccompounds,including chlorophe-nols(VanGestelandMa,1988)polychlorinatedbiphenyls(PCBs), polycyclicaromatichydrocarbons(PAHs)andlinearalkylbenzenes (LABs)(Lamoureuxand Brownawell 1999; Maet al.,1995)was dependentonitsconcentrationintheinterstitialaqueousphase. Furthermore, studies by Savard et al. (2010) have shown that chemicalsmaybeappliedatloadingratesthat exceedaqueous solubility,whichresultedindistortedBSAFvalues.Thispotential complicationwasavoidedinthepresentstudiesbyusingaqueous exposures of the earthworms to test compounds below their aqueous solubility under the test conditions. Other potential complicationscanalsoarise fromfailuretomaintaina constant exposure or dosage, as the source concentration typically decreasessignificantlyoverthecourseoftheuptakeexperiment (Lordetal., 1980).Weaddressedthisissuebyusingastaticaqueous exposuretestsystemwithperiodic(every2-3days)renewalofthe test solution, which ensured consistent chemical exposure conditions duringeachstudy.Additionally,thepotential impor-tanceofbiotalipidcontentindeterminingBCF valueshasbeen notedinpreviousstudiesviavariouslogKOW–logBCFcorrelations
(Lordetal.,1980;ConnellandMarkwell1990;Belfroidetal.,1993). Reviewingthebioconcentrationmeasurementsfororganic com-pounds persistentintheenvironment,Connellcommentedthat thecoefficientforlogKOW,whichwasconsideredasequivalentto
logKLipidW,shouldbethelipidcontentoftheorganismratherthan
a conveniently fitted parameter (Connell 1988; Connell and Markwell, 1990). Consequently, we included earthworm lipid contentmeasurementinourexperimentaldesignandusedthese datafordetermininglipid-basedBCFvalues.Overall,thefeatures ofexperimentaldesigninourpresentstudieshaveallowedusto resolve complex technical challenges, described above, and facilitated thedetermination of the BCF values for4-NAN, 3,5-DNoTAME, and 2-M-5-NPYNE that will contribute to the BCF databasebeingdevelopedforuseinmodelsaimedatpredicting environmentallysignificant parametersfor newmunition com-poundsinsoil.
4.Conclusions
Bioconcentration potentials of new and emerging munition compoundsthat partitionintoearthworms weredeterminedin independent aqueous exposure media studies. These studies showed that the earthworm Eisenia andrei can survive in a sand-Römbkeexposuremediumfor28d,andallowdetermination ofBCFvalues.Thepresentbioconcentrationstudiesweredesigned toseparatethefactorsthatarisefromsoil-waterpartitioningfrom
Table2
Summaryof4-nitroanisole(4-NAN),3,5-dinitro-o-toluamide(3,5-DNoTAME),and2-methoxy-5nitropyridine(2-M-5-NPYNE)uptakekineticsindependentlydetermined usingtheexposureofEiseniaandreiinthesand-liquidstructuredaqueousmedium.
Compound Uptake[C]ss mg/kgdry tissue Media [C]aqss mg/L BCFssa mL/gdry tissue %Lipid glipid/gdry tissue LipidbasedBCFss mL/glipid 4-NAN 1251 26.6 47 14.2 332 3,5-DNoTAME 64.6 10.6 6 10.5 58 2-M-5-NPYNE 94.7 8.7 11 12.1 90 aBCF
ssisbasedontheratioofcompounduptakeconcentrationatsteady-state[C]ssintissuecomparedtoaqueousexposuremediaconcentration([C]ss/[C]aqss).
thatoforganism-exposure,thusavoidingthepotential confound-inginfluenceofsoilcharacteristicsontheuptakedata.Thiswas especiallyimportantfordatadevelopmentforaBCFmodelthatis applicabletoawide rangeofmunition compoundswhichhave nitrogroupsthatbindstronglytotheclayfractionofthesoil.The BCF values determined in the present studies will contribute substantially to the BCF database being developed for use in modelsaimedat predictingenvironmentallysignificant param-eters for new munition compounds in soil. The corresponding estimatesofthelipidbasedL-BCFsswilladvancecomprehensionof
theorganismfactorsthatdirectlyaffectbioconcentration.Together these factors are important for the assessment of new and emerging munition compounds as theyare evaluatedfor their potentialmanufactureanduse,andcorrespondingreleaseintothe environment.
Acknowledgments
TheauthorsthankRosalinaGonzálezForerofromUniversityof Delaware for analytical determinations of test chemicals in exposuremedia,MarkGueltaandVanessaL.FunkfromUSArmy EdgewoodChemicalBiologicalCenterforhelpwithfreeze-drying theearthworms.Thisprojectwascompletedincooperationwith andwith fundingbytheStrategic EnvironmentalResearch and DevelopmentProgram(Arlington,VA,USA).
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