bioconcentration and bioavailability of organotin compounds: influence of ph and humic substances

Bioconcentration and Bioavailability ofOrganotin Compounds: In¯uence of pH andHumic SubstancesPeter W. Looser, Stefan Bertschi and Karl Fent*Swiss Federal Institute for Environmental Science and Technology (EAWAG) and Swiss Federal Instituteof Technology (ETH Zu¨rich), Ueberlandstrasse 133, CH-8600 Du¨bendorf, Switzerland

Bioconcentration of triphenyltin (TPT) andtributyltin (TBT) was studied in the freshwaterorganismsDaphnia magna(zooplankton), Chir-onomus riparius (sediment organism) andThy-mallus thymallus (fish yolk-sac larvae). TPTbioconcentration factors (BCFs) at pH 8 werehighest for Thymallus (2200), followed byChironomus (680) and Daphnia (190). Thedifferences could not be fully explained bydifferent total lipid contents. Metabolism andlower bioconcentration were observed for TBTin Chironomus. The BCFs of both TBT and TPTwere higher at pH 8 than at pH 5, but thedifference was much less pronounced thanpredicted by the octanol–water partition model.This suggests that, besides the hydroxide species(TBTOH and TPTOH), the cations (TBT� andTPT�) are also taken up by the organisms tosome extent and that the octanol–water partitionmodel underestimates the uptake of the chargedspecies. Low concentrations of humic substances(HS) led to small reduction in the bioconcentra-tion of TPT in Daphnia and Thymallus, and asignificant reduction occurred at relatively highconcentrations of HS (>10 mg C lÿ1). The resultsof this study provide an important basis forfuture investigations aiming at a better under-standing of the bioavailability and fate of TBTand TPT in freshwater ecosystems.# 1998 JohnWiley & Sons, Ltd.Appl. Organometal. Chem.12, 601–611 (1998)

Keywords: tributyltin; triphenyltin; bioavaila-bility; bioconcentration factors; humic sub-stances; pH dependence; speciationReceived 5 December 1997; accepted 12 February 1998

INTRODUCTION

Organotin compounds are among the most hazar-dous pollutants in aquatic ecosystems.1 Tributyltin(TBT) and triphenyltin (TPT) are of particularinterest because of their widespread use as biocides.TBT is an important biocide in antifouling paints.The ecotoxicological hazards associated withTBT2–7 have resulted in restrictions on its use inmany countries. In spite of these regulations, therelease of TBT into aquatic ecosystems persists dueto its use in antifouling paints on large vessels, paintremoval from pleasure boats, its application inwood preservation and its resuspension fromcontaminated sediments.8–11 Although TPT hasbeen used as co-toxicant with TBT in someantifouling paints, the major application of TPT isin agriculture.12 TPT is used as a fungicide forvarious crops and enters aquatic ecosystems mainlyvia leaching and run-off from agricultural fields.Tetrabutyltin (TeBT) occurs as a by-product of theproduction of other organotin compounds.Although the amounts of TeBT released intoaquatic environments are globally of minor im-portance, some TeBT-contaminated areas havebeen described.13 The ecotoxicological conse-quences of this contamination, however, are notknown.

With respect to the ecotoxicology of organotincompounds, many studies have been performed onthe contamination of aquatic systems and thetoxicity.1–7,14,15Knowledge has also been gainedon the distribution pattern of organotins in environ-mental compartments.12,16–19 However, the long-term ecotoxicological effects of organotin con-taminants on the structure and function of aquaticecosystems are still not well understood, particu-larly with respect to biomagnification in foodwebs12,20–22 and toxicity of contaminated sedi-ments. These hazards are dependent on thebioavailability of the contaminants.

APPLIED ORGANOMETALLIC CHEMISTRY, VOL. 12, 601–611 (1998)

# 1998 John Wiley & Sons, Ltd. CCC 0268–2605/98/090601–11 $17.50

* Correspondence to: K. Fent, Department of Chemistry, EAWAG,Ueberlandstrasse 133, CH-8600 Du¨bendorf, Switzerland.E-mail: [email protected]

The bioavailability of environmentalchemicalscanbestudiedby assessingthebioconcentrationbymeans of bioaccumulationexperiments.Severalgeneralfactors,suchashydrophobicity,speciationand organism-specificproperties, determine thedegreeof bioconcentration.For neutral organiccompoundsof medium hydrophobicity and lowbiotransformationpotential, octanol–waterparti-tion coefficients(Kow) canserveasan estimateofthe maximumpossiblebioconcentrationin organ-isms.23–25 For chemicalsthat can form chargedspeciesin water, differential bioconcentrationofthe charged and unchargedspecies should beexpected.In many cases,however,simple parti-tioning of the compound between the aqueousphase and the organisms cannot be assumed.Species-specificfactorssuchas uptakeefficiency,growth dilution, metabolism and excretion canmodify thedegreeof bioconcentration.

Organotin compounds,in particular TBT andTPT, differ from neutral organic compoundsintheir properties.The speciationof TBT and TPTshowsa strongpH dependence.26 Both compoundsarepresentascationsat low pH andashydroxidesat higher pH. Although they can also formcomplexeswith other anions, these speciesareonly of minor importance in typical freshwatersystems.Therefore, only the hydroxide species(TBTOH/TPTOH) and the cations(TBT�/TPT�)haveto be considered.The two species,however,exhibit very different partitioning and sorptionbehavior. The octanol–waterdistribution ratios(Dow) of TBT andTPT aremore thanan orderofmagnitudehigher at pH 8 than at pH 3.26 This isrelatedto thefact thatTBTOH andTPTOH,butnotTBT� andTPT�, readily partition into theoctanolphase.26 For partitioning and sorption into humicsubstances(Aldrich HS),bothTBT andTPThaveamaximumdistributionratio DOM of approximately140000l kgOM

ÿ1 betweenpH 5 and6.27 At typicalambient conditions (pH 8), the DOM values areconsiderablylower for bothTBT andTPT (10 000–15000 l kgOM

ÿ1).27

In a previous study, TBT bioconcentrationinDaphniamagnawasfoundto behigheratpH 8 thanat pH 6.28 In the same study, Aldrich humicsubstance(HS) reducedthe bioconcentrationofTBT in Daphnia magna. In the presentwork, wehaveextendedour investigationson the bioavail-ability of organotinsto different freshwaterorgan-isms and different compounds.The major goalswere

(1) to comparethe bioconcentrationof TPT by

three different organismsrepresentingdif-ferentecologicalniches,

(2) to verify whetherthe pH-dependentspecia-tion of TBT and TPT results in differentbioconcentrationfactorsat higherandlowerpH, and

(3) to study the effect of relatively low con-centrations(< 10mg lÿ1) of Aldrich humicsubstance(HS) on the bioconcentrationofTPT.

We report experimentallydeterminedbiocon-centration factors for TPT in Daphnia magna(zooplankton), Chironomus riparius (sedimentorganism)and Thymallusthymallus(fish yolk-saclarvae).Bioconcentrationof TBT, TPT andTeBTin Chironomus riparius at pH 8 and pH 5 iscompared,using TeBT as a referencecompoundthat may undergoonly hydrophobicpartitioning.Finally, resultson the bioconcentrationof TPT inDaphniaandThymallusin thepresenceof Aldrichhumic substance(HS) arepresented.

MATERIALS AND METHODS

Chemicals

Tributyltin chloride TBTCl (>97%), dibutyltindichloride DBTCl2 (�97%), triphenyltin chlorideTPTCl (>97%) and tetrabutyltin TeBT (�98%)were obtained from Fluka Chemie AG (Buchs,Switzerland). Diphenyltin dichloride DPTCl2(>98%) and tripropyltin chloride (�98%) werepurchasedfrom ABS (Basel,Switzerland).Butyltintrichloride MBTCl3 (�95%), phenyltin trichlorideMPTCl3 (�98%)andtripentyltin chloride(�96%)were suppliedby Aldrich (Steinheim,Germany).Solutionsof ethylatedbutyl- and phenyltin com-poundsin methanol(�100mg Sn lÿ1) werestoredin the dark at �4 °C as primary standards.Forexternal calibration, the primary standardswerediluted with hexane. Internal standards wereprepared by diluting tripropyltin chloride andtripentyltin chloride with acetone.Stock solutionsof tributyltin chloride, triphenyltin chloride andtetrabutyltinin acetonewereusedto spikeexperi-mentalmedia.

Aldrich humicsubstance(HS)assodiumhumatewasusedwithout furtherpurification.HS is similarto humicsubstancesin manywetlands.29 AlthoughHS may not exactlyrepresentthe dissolvedhumicandfulvic acidsof manyfreshwaterecosystems,it

# 1998JohnWiley & Sons,Ltd. Appl. Organometal.Chem.12, 601–611(1998)

602 P. W. LOOSER,S. BERTSCHIAND K. FENT

is appropriatefor investigationsof the generalbehavior of dissolvedhumic substancesand theresults can be compared with those of otherstudies.28,30,31,32,33A HS stock solution for theexperimentswasobtainedby dissolving2.5g HSin5 l of nanopurewater, centrifugingat 8000 g for30min andfiltering througha 0.45mm filter.

Daphnia experiments

Daphnia magnawere cultivated accordingto theOECDGuideline20234 in syntheticmediumM735

and fed with the green algae Scenedesmussub-spicatus. The major cations in this medium areCa2� (2 mM), Mg2� (0.5mM) and Na� (0.8mM),while the most important anions are HCO3

ÿ

(0.8mM), SO42- (0.5mM) and Clÿ (4 mM). The

ionic strength of the medium is 13.5mM. TheDaphnia were cultured in a 20� 1 °C climatechamber with a 16h:8h light:dark cycle. Theexperiments were conducted under the sameconditionsusing21�2-day-oldDaphnia that werenot fed during the experiment.Table 1 gives anoverview of the experimentalconditions.In eachtest,80 Daphniawereexposedin 1 l glassbeakers,containing800ml of test solution with TPT. ThepH was adjustedto 8.0 at the beginning of theexperimentwith 0.5M NaOH, monitoredtwice aday,andreadjustedwith 0.5M NaOHif necessary.Beakerswere not aerated.Oxygensaturationwasmeasuredat the beginning and end of eachexperimentandrangedfrom 78.4 to 86.6%.After0, 8, 24,48 and72h, 10 individualswereremoved.TheseDaphnia were rinsedwith nanopurewater,driedoncellulosefilters,weighedandtransferredtoglassvials for storageat ÿ20°C until analysis.At0 h and72h,aliquotsof experimentalmediumweretakenfrom eachglassbeaker,acidifiedto pH 2 andstoredat 4 °C for organotinanddissolvedorganiccarbon(DOC) analyses.In the experimentswithhumic substance(HS), aliquots of the HS stocksolution were addedto the experimentalmedium,resultingin DOC concentrationsof 1.1–14.2mg Clÿ1. Three replicateswere made for every DOCconcentration.

Chironomus experiments

Egg massesof the non-biting midge Chironomusriparius were receivedfrom SpringborneLabora-tories AG (Horn, Switzerland).The larvae wererearedasdescribedby StrelokeandKopp36 in 10 lall-glass aquaria, containing precleaned andshreddedpaper towels as substrateand synthetic

medium M735 as overlying water. The watercolumn was aeratedat a rate of 1–2 bubblespersecondand the larvaewere fed with the fish foodTetramin1. The experimentswereconductedin aclimate chamber at 20� 1 °C with a 16h:8hlight:darkcycle.Eighty two-week-oldChironomuslarvaewereexposedin 1 l glassbeakerscontaining800ml of M7 medium with TBT, TPT or TeBT(Table 1). The larvae were not fed and thecontainerswerenotaeratedduringtheexperiments.Oxygensaturationwasdeterminedat thebeginningandat theendof theexperiments.Themeanvalueswere 67.1� 5.7% and 64.6� 4.3%, respectively.The pH was measuredthree times a day andadjustedto the desiredvaluesof 8.0 and5.0 with0.5M NaOH and1.0M HCl, respectively.After 6,24, 48 and72h, samplesof 10 Chironomuslarvaewere removed, rinsed, dried on filter paper,weighed into vials and stored at ÿ20°C fororganotin analysesand lipid determination.TheTBT and TPT experimentswere replicated fivetimesandtheTeBT experimenthadsix replicates.

Fish larvae experiments

Fertilized eggsof graylings,Thymallusthymallus,from the River Rhine were transportedto thelaboratoryandadaptedto aeratedgroundwaterin aclimatechamberat 15� 1 °C in the dark for threedays.The groundwaterhada total hardnessof 6.8meq lÿ1 (Ca2� �Mg2�), an alkalinity of 5.8 meqlÿ1 and an ionic strength of 14.9mM. DOC,organotins,nitrite and ammoniawere not presentat detectablelevels. Experimentswere performedwith freshly hatchedyolk-sac larvae in the sameclimate chamber, in the dark. In 10 l all-glassaquaria,250larvaewereexposedfor up to 168h in3 l of groundwaterwith TPT (Table1). The larvaldensitywas1.3 – 2.1g lÿ1. Aquariawereaerated,andO2 andpH determineddaily. The averagepHwas8.3� 0.1 andall measuredoxygensaturationvalueswere� 97%. No food was provided.Theexperimentalwater was changedevery 48h, and250ml aliquotswereremovedat the beginningofthe experimentandbeforethe waterwasrenewed.Correspondingwater sampleswere pooled, acid-ified to pH 2 andstoredat4 °C for determinationofDOC and organotins. The experimental set-upincludedonetestwith TPTandnohumicsubstance(HS), and testswith TPT plus 1.0, 1.7, 4.3 and8.8mg Clÿ1 HS. After sevendifferent time points,20–30fish larvaewere removed,rinsed,dried onaluminumfoil, weighedandstoredatÿ20°C until


BIOCONCENTRATIONAND BIOAVAILABILITY OF ORGANOTINS 603

Table 1 Experimentalconditionsa

Organism CompoundExposureconcentration,

cw (mg lÿ1) pHTemperature

(°C)Wet wt(mg)

Dry weight(% wet wt)

Lipid content(% wet wt)

Controlmortality(%)

Daphnia TPT 8.0� 1.4 (15) 8.0 20� 1 2.1� 0.4 (60) 7.1� 0.5 (14) 0.3� 0.1 (14) 20� 11 (15)Chironomus TPT 4.3� 0.5 (5) 5.0 20� 1 5.5� 1.1 (40) 12.0� 1.0 (7) 0.6� 0.1 (7) 3.8� 3.5 (5)Chironomus TPT 4.8� 0.4 (5) 8.0 20� 1 — — — —Chironomus TBT 7.1� 0.8 (5) 5.0 20� 1 4.4� 0.8 (40) — — 9.0� 5.0 (5)Chironomus TBT 6.5� 0.9 (5) 8.0 20� 1 — — — —Chironomus TeBT 1.6� 0.3 (6) 5.0 20� 1 4.5� 0.5 (40) — — 4.9� 2.7 (6)Chironomus TeBT 1.7� 0.6 (6) 8.0 20� 1 — — — —Thymallus TPT 3.2� 0.4 (10) 8.3 15� 1 18.9� 0.9 (40) 21.1� 4.0 (12) 3.1� 0.5 (12) 4 (1)

a Dataaregivenasmean� SD. Numbersin parenthesesindicatethenumberof independentmeasurements.Theexposureconcentration(cw) refersto thegeometricmeanof theconcentrationat the beginningof the experimentand after 72h (Daphnia, Chironomus) or 48h (Thymallus), respectively.—, Not determined.With the exceptionoftetrabutyltin,all exposureconcentrationsreferto therespectivebutyltin andphenyltinchlorides.Thefactorsfor convertingthereportedconcentrationsinto mg Snlÿ1 are0.42,0.39,0.36,0.34,0.39,0.35,and0.31for MBT, DBT, TBT, TeBT, MPT, DPT andTPT, respectively.

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analysis.Theexperimentalconditionsaresummar-ized in Table1.

Organotin analysis

Butyltins (MBT, DBT, TBT, TeBT)andphenyltins(MPT, DPT, TPT) in waterandbiological sampleswere determinedaccordingto methodsdescribedelsewhere,18,37 with slight modifications. Theacidified water samples(pH 2) were transferredinto 50ml volumetric flasks, spiked with theinternal standards tripropyltin and tripentyltinchloride, and extracted with two portions of0.25%tropolonesolution in pentane–diethylether(2 : 3) andoneportion of pentane.The combinedorganic phaseswere dried on anhydrousCaCl2,concentratedto 1 ml underagentlenitrogenstreamandethylatedwith 2M ethylmagnesiumbromideintetrahydrofuran. After 10min, the excess ofGrignard reagent was hydrolyzed by dropwiseaddition of 1 M HCl. The top layer was removedand concentratedunder nitrogen to 2 ml for GCanalysis.Biological sampleswereacidifiedto pH 2with HCl, homogenizedandspikedwith tripropyl-tin and tripentyltin chlorides.The extractionandderivatizationprocedurewasthesameasfor watersamples,but the extractswerepurified by adsorp-tion chromatographyonsilicagel(0.5g of silicagelin a Pasteurpipet) and subsequentelution withhexane.ThehexaneextractswereanalyzedusingaCarlo Erba HRGC 5160 gaschromatographwithsplit/splitlessinjector,a flamephotometricdetector(SSD 250) and a DB-5 column (J&W) with i.d.0.32mm and a 0.25mm film. Recovery ratesrefering to tripropyltin and tripentyltin chloridewereroutinelyassessedin everysampleandrangedfrom 51 to 81% andfrom 55 to 69% in waterandbiologicalsamples,respectively.Therelativelylowoverallrecoveryratesaremainlydueto thefact thatthewholeanalyticalprocedurewascoveredby thisinternal standardization method. The reportedrecoveriesincludereducedextractionefficiencyinwater sampleswith humic substancesas well asslight evaporationlossesin theconcentrationstepsandsorptionto silica gel (phenyltincompoundsinbiological samples). The detection limits forbutyltin and phenyltin compoundsranged from0.09to 0.35mg lÿ1 in watersamplesandfrom 24to139ng gÿ1 in biota. With the exception oftetrabutyltin, all resultsin this paperrefer to therespectivebutyltin andphenyltinchlorides.

DOC analyses

Water sampleswere membrane-filtered(0.45mm)and DOC was determinedby high-temperaturecombustionfollowed by IR detectionof CO2 usinga Shimadzu TOC-5000A total organic carbonanalyzer. In the controls without HS, DOCconcentrationsof 1.5mg C lÿ1 (mediumM7) and1.7mg C lÿ1 (groundwater)were detectedat theend of the experiments. These residues weresubtracted from the measured DOC, as theyoriginate partly from the acetonein the spikedstocksolutionsandpartly from exudatesfrom thetestorganisms.

Determination of total lipid content

Thetotal lipid contentin Daphnia, ChironomusandThymallussampleswas determinedfollowing thesulfophosphovanillinmethodof BarnesandBlack-stock38 with specificadaptationsfor small organ-isms.39 The lipids were extracted from thelyophilized samples with hexane–isopropanol(3:2) and hydrolyzed with sulfuric acid. Afterreaction with vanillin in concentratedorthopho-sphoricacid for 90min, the absorbanceat 528nmwas measuredwith a Hitachi U-1000 UV/Visspectrophotometer.The calibrationstandardswereobtainedby treatingtriolein standardsin the sameway asthesamples.

Data analysis

Theorganotinuptakecurveswerefittedthroughtheexperimental data points with the integratedequationof the one-boxmodel.40,41,42The modelyielded the uptake rate constant k1 and theelimination rate constantk2 as fitted parameters.Individualbioconcentrationfactorswerecalculatedfor eachreplicateandthe differencesbetweenthedifferentpH valuesandthedifferentconcentrationsof humic substancewere tested for statisticalsignificance with a Student t-test (two-sided,significancelevel P = 0.05).

The following definitions and equationswereused:

One-boxmodel, integrated form (Eqn [1])

cb

cw� k1

k2�1ÿ eÿk2t� �1�

wherek1 = uptakerate constant(ml gÿ1 hÿ1); k2 =elimination rate constant(ml gÿ1 hÿ1); cb = con-centrationin biota(nggÿ1); cw = exposureconcen-



tration (nggÿ1 or ngmlÿ1), calculatedas the geo-metric meanof the concentrationat the beginningof theexperimentsandtheconcentrationafter48h(Thymallus) or 72h (Daphnia, Chironomus).

BCF (bioconcentration factor at the end of theexperiment; Eqn [2])

BCF� cb

cw�2�

All BCF values reported in this study wereexperimentallydeterminedandrefer to the endofthe experiments,during which a steadystatewasnot necessarilyachieved.

RESULTS AND DISCUSSION

Bioconcentration at pH 8

The concentrationsof organotinsin the exposurewatersdecreasedin all the experiments.The TPTconcentrationdecreasedin the experimentswithDaphnia from 8.6� 1.7 to 7.4� 1.6mg lÿ1, withChironomusfrom 6.3� 0.4 to 3.1� 0.6mg lÿ1 andwith Thymallusfrom 4.2� 0.2 to 2.4� 0.5mg lÿ1.In the TBT andTeBT experimentswith Chirono-mus, theconcentrationsdeclinedfrom 10.0� 1.0to4.7� 0.8mg lÿ1 andfrom 3.1� 0.5to 0.9� 0.4mg,lÿ1, respectively.Takinginto accounttheorganotinconcentrationsin water and biota, no quantitativemassbalancewasachieved.Lossesmayhavebeencausedmainlyby absorptionto theglasswalls.Theanalysesof watersamplesfrom controlexperimentsshowed,however,that the decreasein the concen-trations occurredin a time-dependent,non-linearfashion(datanotshown).Thus,thegeometricmeanof the measuredinitial and final concentrationsgives realistic values for the averageexposureconcentrations(Table1).

The uptake of TPT was species-dependent.ConsiderabledifferencesoccurredamongDaphnia(pH 8.0),Chironomus(pH 8.0) andThymallus(pH8.3),asshownin Fig. 1. A steadystatewasreachedin the experimentwith Daphnia, whereasbiocon-centration in Chironomus was approaching aplateauafter 72h, but did not reacha steadystate.In Thymalluslarvae, high uptakepersisted,evenafter 168h. The bioconcentrationfactors(BCF) atthe endof the exposureperiodswere190� 50 forDaphnia, 680� 200 for Chironomusand2200forThymallus. Of the major metabolitesof TPT, onlydiphenyltin (DPT), but not monophenyltin(MPT)

Figure 1 Bioconcentrationof TPT in (a) Daphnia magna(n = 3, 30 Daphnia), (b) Chironomus riparius (n = 5, 50Chironomus) and(c) Thymallusthymallus(n = 1,10Thymallus)at pH 8.0 and8.3, respectively.Data are given asmean� SD

except for Thymallus(only one experiment).Also shown isdiphenyltin (DPT) as a possiblemetabolite.Monophenyltin(MPT) wasnot detectablein anyof theorganisms.



could be detectedin the organisms.DPT did notincreasesignificantlyin eithertheorganismsor theexposurewaters during the experiments.For allspeciesthemortality in theexposurewatersdid notdiffer significantly from the control mortality(Table 1). The experimentallydeterminedBCFsand toxicokinetic parametersfor Daphnia andChironomusat pH 8.0 and and for ThymallusatpH 8.3 arelisted in Table2.

SinceDaphnia, Chironomusand Thymallusareorganismswith different physiologicaland ecolo-gical characteristics,the differencesbetweentheBCFsarenot surprising.Partof thedifferencescanbe explainedby the different lipid contents(Table1).Onawetweightbasis,thetotal lipid contentsforDaphnia, Chironomusand Thymallusare 0.3, 0.6and 3.1%, respectivelyi.e. they are in a ratio of

1:2:10. The ratio of the BCFs determined inDaphnia, Chironomus and Thymallus is1:3.6:11.6.It shouldbe notedthat in ChironomusandThymallusasteadystatewasnot reached.Fieldstudieswith various vertebratesreportedthat thedistributionpatternof TBT andTPTin thedifferentorganswas not relatedto the lipid contentof thecorresponding tissues.12,20 It can therefore beassumedthat species-specificfactors other thanlipid content,such as uptakemechanism,toxico-kinetics and other factors accountfor the differ-encesamongspecies.

Figure2 showsthe bioconcentrationof TBT inC. riparius at pH 8.0. A steadystatewasreachedwithin the72h exposureperiod.ForTBT, aBCFof310� 100 was determined at the end of theexperiment. In contrast to the experiment withTPT,significantconcentrationsof metaboliteswereobserved.Dibutyltin (DBT) in thetissuesincreasedduring the experimentand reacheda steadystate(Fig. 2). After 72h, theconcentrationsof theDBTtissueresidueswereabout50%of thoseof theTBTtissueresidues.Monobutyltin(MBT) occurredonlyin low concentrations.The concentrationsof DBTandMBT in theexposurewatersremainedconstantover time at levels of 0.38� 0.09mg lÿ1 and0.12� 0.03mg lÿ1, respectively, indicating thatDBT wasnot releasedby theorganisms.However,the DBT tissueconcentrationsleveledoff quickly(Fig. 2) suggestingthat either TBT metabolismsloweddownafterabout24h or theDBT producedwasmetabolizedfurther.

Basedon thelog Kow valuesfor TPTandTBT atpH 8 (3.5 and 4.1, respectively),a lower BCFwouldbeexpectedfor TPT thanfor TBT. TheTPTand TBT bioconcentrationfactors determinedinChironomuscontrastsharplywith thepredictionsofthe octanol–watermodel.The BCF for TBT (310)

Table 2 Toxicokineticparametersandexperimentallydeterminedbioconcentrationfactorsfor TPT, TBT andTeBTa

Organism CompoundNumberofreplicates

Uptakerateconstant,

k1 (ml gÿ1 hÿ1)

Elimination rateconstant,

k2 (ml gÿ1, hÿ1)

BCF at endofexperiment,

cb/cw

Durationofexperiment(h)

Daphnia TPT 3 9.7� 2.2 0.049� 0.019 190� 50 72Chironomus TPT 5 19.9� 4.0 0.025� 0.009 680� 200 72Chironomus TBT 5 28.6� 8.9 0.102� 0.039 310� 100 72Chironomus TeBT 6 17.1� 9.5 0.002� 0.019 1200� 300 72Thymallus TPT 1 15.1 0.002 2200 168

a Dataaregivenasmean� SD. TheDaphniaandChironomusexperimentswereperformedat pH 8.0,theThymallusexperimentatpH 8.3.Theionic strengthin theexperimentalwaterwas13.5mM for theDaphniaandChironomusexperiments,and14.9mM for theThymallusexperiment.A steadystatewasachievedfor TPT in Daphniaandfor TBT in Chironomus. In theTPT experimentwithChironomus, bioconcentrationwasapproachinga plateauafter72h, but did not reacha steadystate.In theTeBT experimentwithChironomusandin the TPT experimentwith Thymallus, considerableuptakepersistedat theendof theexperiment.

Figure 2 Time course of tissue TBT, DBT and MBTconcentrationsin Chironomuslarvae.Exposureconcentrationwas6.5mg TBT lÿ1 at pH 8.0.Dataaregivenasmean� SD of50 Chironomus(n = 5). The increasingDBT concentrationsindicatemetabolismof TBT.



is lower by a factorof morethantwo thantheBCFfor TPT (680). This result is related to TBTmetabolismin Chironomus, whereasno metabo-

lism of TPT couldbeobserved.Thepresenceof anefficient TBT metabolismin Chironomusis sup-ported by the fact that the DBT tissue residuesincreasedsignificantly over time, reaching highlevels,while the concentrationsof MBT andDBTin the exposurewatersremainedconstantat a lowlevel. Furthermore,achievementof a steadystatewithin threedaysis at leastpartly theresultof TBTmetabolism,which is indicated by the relativelyhigheliminationrateconstantk2 of 0.10� 0.04hÿ1

(Table 2). A short time until achievementof asteadystate(sevendays)hasalsobeenfoundfor theuptakeof TBT by Hyalella azteca,43 but concen-trationsof DBT andMBT werenot reported.Stabetal.12 foundhighconcentrationsof MBT andDBTin chironomidsandGammarusandpointedout thepossibilityof relativelyhighTBT degradationratesin theseorganisms.Our study demonstratesTBTmetabolismin Chironomusriparius, which is ofimportancefor thefateof TBT in thefood websoffreshwaterlakesandrivers,asvariousfish speciesarepredatorsof chironomids.

A muchhigherbioconcentrationof tetrabutyltin(TeBT) than TPT or TBT was observed inChironomus. The experimentallydeterminedBCFat pH 8.0 was1200� 300after72h (Table2). Noother butyltin compoundsoccurredat detectablelevels in Chironomuslarvae in this experiment.Metabolismwas thereforenot present,or only ofminor importance.To our knowledge,no octanol–waterpartitioncoefficient(Kow) for TeBT hasbeenreportedsofar, but it is expectedto behigherthantheKow of TPTOHor TBTOH.

In¯uence of pH

Figure3(a) showsthe bioconcentrationof TBT inChironomuslarvaeatpH 8 andpH 5.A steadystatewasachievedwithin theexposureperiod.TheBCFvaluesdeterminedafter72h were310� 100(pH 8)and170� 30 (pH 5). ThedifferencebetweentheseBCFs is statistically significant (t-test, two-sided,P = 0.045,n = 5). As found at pH 8 (Fig. 2), theDBT residuesin the larvaealsoincreasedat pH 5,to a level approximately50% that of the TBTresidues,indicating metabolism of TBT. Thesefindings are consistentwith the relatively highelimination rate constantsk2 of 0.10� 0.04 hÿ1

(pH 8) and0.12� 0.04hÿ1 (pH 5) comparedwiththek2 valuesin theTPT experiment(Table2). Thebioconcentrationof TPT was also higher at pH 8thanat pH 5 (Fig. 3b), but the differencewaslesspronouncedthanfor TBT andwasnot statisticallysignificant (t-test, two-sided,P = 0.17, n = 5). At

Figure 3 Bioconcentrationof (a)TBT, (b) TPT and(c) TeBTin Chironomuslarvaeat pH 8 andpH 5. TBT andTPT dataaregiven as mean� SD of 50 Chironomus(n = 5), TeBT dataasmean� SD of 60 Chironomus(n = 6).



the end of the experiment, the BCFs were680� 200(pH 8) and510� 120(pH 5).

ThehigherBCFof TBT atpH 8 thanatpH 5 maybe explainedby a higheruptakeof the unchargedhydroxidespeciesascomparedwith the cation.AtpH 8 the hydroxide complex TBTOH predomi-nates.The pKa value of TBT is 6.25.26 Basedonthis pKa, the fractionof TBTOH dropsbelow10%at pH 5, and the cation TBT� becomes thepredominantspecies.TPT has a pKa of 5.2,26

which resultsin approximately40% TPTOH and60%TPT� atpH 5. Theweakereffectof thepH onthe bioconcentrationof TPT, comparedwith TBT,

is consistent with the higher fraction of thehydroxidespeciesat pH 5. This indicatesthat thehydroxide speciesTBTOH and TPTOH are thepredominantforms takenup by theorganism.

Although the uptake of TBT and TPT byChironomuslarvaewashigherat pH 8 thanat pH5, the difference is less pronouncedthan theoctanol–watermodelwould predict.For TBT, theoverall octanol–waterdistributionratios(log Dow)are 4.1 and 2.9 at pH 8 and pH 5, respectively.26

Basedon theseresults,the accumulationof TBTwouldbeexpectedto bemorethantenfoldloweratpH 5 thanat pH 8. Theexperimentallydetermined(steady-state)BCFsdifferedby afactorof only 1.8.Clearly, the octanol–waterpartition model under-estimatesthebioconcentrationof TBT atpH 5. Thediscrepancymayberelatedto differentbehavioroftheTBT� cationsin biological membranesthaninoctanol–watersystems. It has been shown forsubstituted phenolic compounds that chargedspeciespartition to a higherextentinto liposomesthantheoctanol–waterdistributionratios(log Dow)predict.44 It shouldbe investigatedwhetheror nottriorganotincationsbehavein a similar fashiontochargedspeciesof other hydrophobic ionizablecompounds.A further explanationfor the discre-pancy with the octanol–watermodel may be pHdifferencesat theuptakemembranesrelativeto thepH of theambientwater.Althoughthemechanismremainsto be elucidated,the resultsindicate thatChironomuslarvaetake up the cationsTBT� andTPT� more efficiently than the octanol–waterpartition modelsuggests.

Figure 4 Bioconcentrationof TPT at differentconcentrationsof humicsubstances(DOC in mg C lÿ1 in (a) Daphniaand(b)Thymalluslarvae,at pH 8.0 andpH 8.3, respectively.Dataaregiven as mean� SD of 30 Daphnia (n = 3) and valuesfor 10Thymalluslarvae(n = 1), respectively.

Figure 5 Influence of different concentrationsof humicsubstances(DOC in mg C lÿ1 on the bioconcentrationof TPTin (a)Daphniaand(b) Thymalluslarvae.Datareferto themeanbioconcentrationfactors(cb/cw) at 48 and72h in Daphniaandat the endof theexperiment(168h) in Thymalluslarvae.



In the experiment with TeBT (Fig. 3c), theChironomuslarvaeshowedaslightly higherBCFatpH 5 (1500� 650) thanat pH 8 (1200� 310),butthedifferencewasnotsignificant(t-test,two-sided,P = 0.39,n = 6). In contrastto TBT andTPT,TeBTdoes not dissociatein water. Therefore, no pHdependenceof the TeBT speciationwasexpected.Thesimilar BCFsfor TeBT at pH 8 andpH 5 thusindicate that the H� concentrationitself has nosignificant influence on the bioconcentrationatthesepHs. It canbe concluded,therefore,that thedifferencesin thebioconcentrationof TBT andTPTat pH 8 and pH 5 are related to the chemicalspeciationratherthanto pH-inducedalterationsofuptakemembranes.

In¯uence of humic substance

Figure4(a) showsthe bioconcentrationof TPT inDaphniamagnain the presenceof Aldrich humicsubstance(HS). The HS concentrationsrangedfrom 0 to 14.2mg C lÿ1 and the pH was8.0. ForDaphniain exposurewaterswithout HS, a BCF of190� 50 was determined.The BCFs for experi-mentswith 1.1, 4.4, 8.0 and 14.2mg C lÿ1 were150� 60,100� 40,120� 40and60� 30,respec-tively. The averagemortality underTPT exposure(33� 12%, n = 15) was not significantly differentfrom the control mortality (20� 11%, n = 15). Asignificant decreasein the BCF was only obser-vablefor anHS concentrationof 14.2mg C lÿ1 (t-test, two-sided, P = 0.03, n = 3). Lower DOCconcentrationsdid not producesignificanteffects.A very similar picturewasobtainedin experimentswith fish yolk-saclarvae,Thymallusthymallus, atpH 8.3 (Fig. 4b). The BCFs at the end of theexperimentswere2240(0 mgC lÿ1), 1900(1 mgClÿ1), 1550(1.7mg C lÿ1), 1520(4.3mg C lÿ1) and2000 (8.8mg C lÿ1). Although there is a trendtowards lower BCFs in the presenceof HS, thedifferences between the determined BCFs aremodestandmay in partbeattributedto thenaturalvariability of the fish yolk-sac larvae. Meanmortality in all testswith TPT (3.5� 3.1%,n = 5)was not significantly different from the control(4%). It shouldbenotedthata steadystatewasnotreachedwithin the168h experimentalperiod.

A variety of studies have shown that thebioavailability of hydrophobicorganic chemicalsis reduced in the presence of humic sub-stances.30–33,45 Similar effects were shown forTBT in a previous study with Daphnia andThymallus.28 It cangenerallybeexpectedthathighconcentrationsof humic substancesreduce the

bioavailabilityof organotins.Concentrationshigherthan10mg C lÿ1 are,however,not very represen-tative for many natural freshwatersystems.Sincethe DOM value of TPT is approximately15 000 lkgOM

ÿ1 for Aldrich humic substance(HS) at pH8,27 a concentrationof 10mg C lÿ1 is expectedtoreducethe free TPT concentrationby a factor of1.3.LowerHSconcentrationswill resultin aminorreduction of the bioavailable TPT fraction. Theresults of the experiments with Daphnia andThymallus lead to the hypothesisthat relativelyhigh HS concentrations(>10mg C lÿ1) areneces-sary to generatepronouncedeffects on the bio-availability of TPT to freshwaterorganisms(Fig.5).

CONCLUSIONS

This study showsdifferencesin the bioaccumula-tion of TPT in three different freshwaterspeciesrepresentingdifferentecologicalniches.Total lipidcontentexplainsa partof thedifferences,but otherspecies-specificfactors such as toxicokineticsinfluencethebalancebetweenuptakeandelimina-tion. Rapid achievementof a steadystatemakesChironomus riparius a promising species formonitoringTBT andTPT availability in contami-natedharborsediments.Furthermore,Chironomusis akeyorganismin benthiccommunities.Thus,thepresence of an efficient TBT metabolism inChironomuslarvae can influencethe distributionpatternof butyltin compoundsin the food websoffreshwaterlakes. If additional organismsat lowtrophiclevelscanmetabolizeTBT moreeasilythanTPT, a lower biomagnificationpotential can beexpectedfor TBT thanfor TPT.

Both thepH andhumicsubstancesinfluencethebioconcentrationof organotinsin freshwaterorgan-isms.The bioconcentrationof TBT and TPT wasfound to be higher at the ambientpH of surfacewaters than at low pH. The hydroxide speciesTBTOH and TPTOH are taken up to a higherdegreethanthecorrespondingcations.Thecations,however, are more readily taken up than theoctanol–watermodel would suggest.Humic sub-stances(HS) reducethebioavailabilityof TBT andTPT, but only relativelyhigh concentrationsof HSlead to a substantialreduction. In contaminatedharbor sediments,however,the bioavailability ofTBT or TPTmaybereducedby highorganicmattercontents.

Further experimentswith Chironomusriparius



areneededto answertheopenquestionsconcerningorganotinmassbalancesandto providequantitativetoxicokineticdata.Futureresearchshouldfocusonthebioavailability andtoxicity of TBT andTPT insediments.

Acknowledgments We thankV. Wuthrich, RCC Itingen, forprovidingDaphnia, J. vanderKolk, SpringborneLaboratories,for eggmassesof Chironomus, J.Walterfor eggsof Thymallus,G. Meier for helpwith lipid determinations,andC. Arnold, M.Clayton,B. Escher,S.Muller andR.Schwarzenbachfor helpfuladviceandfor reading themanuscript.

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bioconcentration and bioavailability of organotin compounds: influence of ph and humic substances

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