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Ion trapping of amines in protozoa – a novel removal1
mechanismformicropollutantsinactivatedsludge2
3
RebekkaGuldea,SabineAnlikera,b,Hans-PeterKohlera,b,KathrinFenner*a,b,c4
5aEawag,SwissFederalInstituteofAquaticScienceandTechnology,8600Dübendorf,Switzerland6bInstituteofBiogeochemistryandPollutantDynamics,ETHZürich,8092Zürich,Switzerland7cDepartmentofChemistry,UniversityofZürich,8057Zürich,Switzerland8
9
*Correspondingauthor:[email protected],Tel.:+4158765508510
11
Wordcount:601312
Tables(2small)+Figures(2small,1large):180013
Totalwordcount:781314
15
16
Abstract17
Tooptimizeremovaloforganicmicropollutants fromthewatercycle,understandingtheprocesses18
during activated sludge treatment is essential. In this study,wehypothesize that aliphatic amines,19
which are highly abundant amongst organic micropollutants, are partly removed from the water20
This document is the accepted manuscript version of the following article:Gulde, R., Anliker, S., Kohler, H. -P. E., & Fenner, K. (2018). Ion trapping of amines in protozoa: a novel removal mechanism for micropollutants in activated sludge. Environmental Science and Technology, 52(1), 52-60. http://doi.org/10.1021/acs.est.7b03556
2
phase in activated sludge through ion trapping in protozoa. In ion trapping, which has been21
extensively investigated in medical research, the neutral species of amine-containing compounds22
diffusethroughthecellmembraneandfurtherintoacidicvesiclespresentineukaryoticcellssuchas23
protozoa.Theretheybecometrappedbecausediffusionofthepositivelychargedspeciesformedin24
the acidic vesicles is strongly hindered.We testedour hypothesiswith two experiments. First,we25
studied the distribution of the fluorescent amine acridine orange in activated sludge by confocal26
fluorescence imaging.Weobserved intensefluorescence indistinctcompartmentsoftheprotozoa,27
butnotinthebacterialbiomass.Second,weinvestigatedthedistributionoftwelveamine-containing28
and eight controlmicropollutants in both regular activated sludge and sludgewhere theprotozoa29
hadbeeninactivated.Incontrasttomostcontrolcompounds,theamine-containingmicropollutants30
displayedadistinctlydifferentbehaviorinthenon-inhibitedsludgecomparedtotheinhibitedone:i)31
moreremovalfromtheliquidphase;ii)deviationfromfirst-orderkineticsfortheremovalfromthe32
liquidphase;and iii)higheramounts inthesolidphase.Theseresultsprovidestrongevidencethat33
iontrappinginprotozoaoccursandthatitisanimportantremovalmechanismforamine-containing34
micropollutants in batch experiments with activated sludge that has so far gone unnoticed. We35
expect thatour findingswill trigger further investigationsonthe importanceof thisprocess in full-36
scalewastewatertreatmentsystems,includingitsrelevanceforaccumulationofammonium.37
38
Introduction39
Manycompoundsineverydayuse,suchaspharmaceuticals,personalcareproducts,surfactantsand40
biocides,areconveyedbysanitarysewerstowastewatertreatmentplants(WWTPs),wheretheyare41
removedtodifferentextents.1Thetreatmentstagemainlyresponsibleforremovaloftheseso-called42
organicmicropollutants (MPs) inWWTPs is activated sludge treatment, duringwhichMPsmaybe43
removedbydifferentprocesses, includingabiotic transformation, sorption to the sludge flocs, and44
microbial biotransformation.1-4 Different classes of wastewater-relevant MPs contain structural45
motifs that include a basic functional group. Particularly among the active ingredients of46
pharmaceuticals, basic functional groups have been reported to be present in about 40% of the47
structuresandtobedistributedacrossmanytherapeuticclasses.5, 6Amongthese,aliphaticamines48
withaciddissociationconstants (pKa) in the rangeof7 to10aremostabundant.5Their speciation49
changesatenvironmentallyrelevantpHvalues,suchthatunderratheracidicpHconditionsaliphatic50
amines are typically protonated and hence cationic, while they become deprotonated and hence51
neutral at higherpH. In aprevious study,we showed that, due to their basicity, amine-containing52
MPsexperiencepH-dependentbiotransformationinactivatedsludgecommunities.7Specifically,we53
observedthatremovalofaminesfromtheliquidphaseproceededfasterathigherpHvalues,which54
wasexplainedbythefactthattheneutralspeciescanpassivelydiffusethroughthecellmembranes,55
4
byenergy-dependentprotonpumps.11Asaconsequence,theamine-containingcompoundsbecome82
positively charged, and, since the charged form is hindered from diffusing back into the cytosol,83
becomeeffectivelytrappedinsidethevesicles.Diffusionintothevesiclescontinuesuntilequilibrium84
of the neutral species between the extracellular environment, the cytosols and the vesicles is85
reached. Due to the considerable pH difference between the extracellular environment and the86
vesicles, this can lead to several orders of magnitude difference in total amine concentrations87
between the extracellular environment and the vesicles.12 Consistent with this mechanism, ion88
trapping has been described to increase with increasing pKa and lipophilicity of the drugs and at89
elevatedextracellularpH.12,15Iontrappinginacidicvesicleshasbeendescribedacrossawiderange90
ofeukaryoticcells,fromyeasttoanimalcells.12,13Inmedicalresearch,iontrappingisevenusedfor91
staining acidic vesicles with fluorescent amines such as LysoTracker red, quinacrine, and acridine92
orange16, which can subsequently be detected by means of confocal fluorescence imaging. 6, 1693
Especiallyacridineorangeisahighlyversatiledye,whichevenallowsdifferentiatingbetweenacidic94
vesicleswithdifferentpH.Uponexcitation,itemitsgreenlightatlowconcentrationsand,duetothe95
formation of stacks of molecules, red light at high concentrations. 16 It is noteworthy that ion96
trapping goes beyond the effect of extracellular pH on bacterial toxicity that has previously been97
described for speciating chemicals, including ammonia. 17-19 Themagnitude of this latter effect is98
typicallysufficientlyexplainedbythepH-differencesbetweenextra-and intracellularpH17without99
theneedtoinvoketrappinginacidicvesicles,whichareabsentfrommostbacteriaanyway.100
At this point, we hypothesized that protozoa, whichmake up about 10% of the activated sludge101
biomass,20 could be important for the fate of amine-containingMPs in sewage sludge treatment.102
Since protozoa are eukaryotes, they posses acidic vesicles which can trap amine-containing103
compounds. Indeed, it has previously been demonstrated through staining experiments that the104
protozoic ciliateTetrahymena thermophilawas able to accumulate the fluorescent amine acridine105
orange.21Protozoainactivatedsludgemainlybelongtooneoffivegroups,namelyamoeba,ciliates106
(free-swimmingandstalked),flagellates,suctoreansandrotifers,whicharemulti-celledorganisms.20107
Therefore, the goal of this study was to test the hypothesis that ionizable amine-containing108
compoundsare trapped in theacidicvesiclesof theprotozoiccommunityofactivatedsludge.This109
would constitute a previously unknown, additional removal process for amine-containing MPs in110
activated sludge. To test this hypothesis, we conducted two kinds of experiments. First, we111
investigated the accumulation of the fluorescent amine acridine orange in an activated sludge112
community by confocal fluorescence imaging. Second, we examined the distribution of non-113
fluorescent MPs in the liquid and solid phase of activated sludge and compared it to conditions114
wheretheprotozoawereinactivatedbytheadditionoftheinhibitordigitonin.20Theseexperiments115
5
wereconductedwith12amine-containingtargetMPsandeightcontrolMPswitheitherneutralor116
neutral-anionicspeciation.117
118
MaterialsandMethods119
Thefollowingisacompendiouspresentationofthematerialsandmethods;fulldetailsaregivenin120
theSupportingInformation(SI).121
MicropollutantSelection122
Altogether, experimentswere carriedoutwith20environmentally relevantMPs. The following12123
amine-containingMPsthatundergocationic-neutralspeciationwereselectedastargetcompounds124
(atenolol, ranitidine, venlafaxine, lidocaine, tramadol, levamisole, mexiletine, fenfluramine,125
citalopram,propranolol,mianserin, ticlopidine).ThreeMPsthatundergoneutral-anionicspeciation126
(sulfathiazole, naproxen, trinexapac-ethyl) and five MPs that remain predominately neutral127
(diethyltoluamide, alachlor, azoxystrobin, isoproturon, chlortoluron) were selected as control128
compounds.Abbreviations(ID),chemicalstructures,andpredictedpKavaluesarepresentedinTable129
1. Additionally, separate experimentswere conductedwith the fluorescent amine acridine orange130
(AO)alsolistedinTable1.131
132
Table1:CompoundID,CompoundName,Structure,ChargeStateofIonizedSpeciesintheRelevantpHRange133
(i.e.,pH4-8),andPredictedpKaValues.134
ID Name Structure ChargeStatestate
pKaa
SUL Sulfathiazole
anionic 6.9
NAP Naproxen
anionic 4.2
TRI Trinexapac-ethyl anionic 3.4
DET Diethyltoluamide
neutral
ALA Alachlor
neutral
AZO Azoxystrobin
neutral
ISO Isoproturon
neutral
CLT Chlortoluron neutral
ATE Atenolol
cationic 9.7
6
RAN Ranitidine
cationic 7.8
VEN Venlafaxine cationic 8.9
LID Lidocaine
cationic 7.8
TRA Tramadol cationic 9.2
LEV Levamisole
cationic 7.0
MEX Mexiletine
cationic 9.5
FEN Fenfluramine
cationic 10.2
CIT Citalopram
cationic 9.8
PRO Propranolol cationic 9.7
MIA Mianserin
cationic 6.9
TIC Ticlopidine
cationic 6.7
AO AcridineOrange
cationic 8.2
apKavaluesaspredictedby22135136
Experimentswithacridineorange137
Reactors (100mLamber Schott bottles)were filledwith50mLactivated sludge sourced from the138
nitrificationbasinof a full-scale SwissWWTP (diluted to a total suspended solids concentrationof139
approximately 1 g/L) and shaken at 160 rpm on a circulating shaker table to ensure continuous140
mixing and aeration. Triplicate reactors were spiked with 60 µL AO solution (50 mg/L in141
methanol:water1:9),resultinginafinalconcentrationofabout60µg/L.Afteratimeperiodof26to142
29.5 h, samples were investigated with a Leica SP5 Laser Scanning Confocal Microscope (Leica,143
Heerbrugg, Switzerland)with an excitationwavelength of 458 nm and an emissionwavelength of144
480-560nmformonomersemittinggreenlightatlowconcentrations,and590-660nmforstacksof145
AOemittingredlightathighconcentrations.146
147
ExperimentswithselectedMPs148
In activated sludge, removal of MPs from the liquid phase can in principle be caused by abiotic149
transformation, sorption to sludge, ion trapping, andmicrobialbiotransformation. Ingeneral,both150
7
ion trapping and microbial biotransformation might involve bacterial and protozoic parts of the151
community.Totest thehypothesis thatprotozoic iontrapping isan importantremovalprocess for152
amines, the following experimental setupwas chosen. (i) Control experimentswere conducted to153
assess the magnitude of abiotic transformation and sorption processes (see below for details on154
these experiments). (ii) Experiments were carried out with the addition of digitonin. Digitonin155
inactivatestheprotozoaoftheactivatedsludgecommunitybyformingcomplexeswithcholesterol156
that are largeenough to inducepermanentholes ineukaryoticmembranes 23. Since cholesterol is157
present ineukaryoticbutnotbacterialcellmembranes,additionofdigitonin isexpectedto leadto158
selectivefunctionalinactivationorevendestructionofprotozoiccellsinactivatedsludge.Therefore,159
throughcomparisonoftheexperimentscarriedoutunderinhibitingandnon-inhibitingconditionsfor160
protozoa, the contribution of protozoic and bacterial processes, which could include both ion161
trappingandbiotransformation,couldbedifferentiated.Toverifythattheadditionofdigitonindid162
not affect other processes than the protozoic ones, experimental conditions (i.e., pH, total163
suspended solids concentration, oxygen uptake rate, ammonia uptake rate, and nitrate formation164
rate)wereclosely followedandcomparedbetween inhibitingandnon-inhibitingconditions. (iii)To165
differentiate ion trapping from biotransformation, the fate of the 12 target amines and the eight166
controlMPswasassessedaccordingtothreespecificcriteriaindicativeofiontrappingasfollows:167
• Concentration inthe liquidphase:Since, inthecaseof iontrapping,concentration levels in168
the liquid phase are expected to be lower under non-inhibiting than under inhibiting169
conditions, the differences between themean concentrations under these two conditions170
were calculated for each time point. The time point with the maximal concentration171
differencewasselectedforevaluation.Differencesof>8.7µg/L(givenaspikeconcentration172
of60µg/L,fordetailsseebelow)wereconsideredsignificantsincethisvaluecorresponded173
to the average maximal difference between concentrations of replicate samples for all174
compoundsanalyzed.175
• Removalkineticsintheliquidphase:PlotsoftheconcentrationofMPsasafunctionoftime176
fortheexperimentscarriedoutundernon-inhibitingconditionsareexpectedtodeviatefrom177
first-orderkinetics.Thisisbecausetheiontrappingprocess,whichshouldeventuallyleadto178
the establishment of equilibrium across the different membranes, needs some time to179
establish,butalsocoincideswithbiotransformation.Thepresenceorabsenceoffirst-order180
kinetics was evaluated by means of visual inspection and R2 of the linear fit to the181
logarithmizeddata.However, since theevaluationof removalkinetics isonlymeaningful if182
significantremovaltakesplaceatall,thisevaluationwasonlyconductedforcompoundswith183
a removal rate constant > 0.1 d-1. Under protozoa-inhibiting conditions, where first-order184
kineticsareexpected,allcompoundsmeetingtheaboveremovalratecriteriondisplayedR2185
8
values > 0.88 (see Table S8). Therefore, this value was chosen as a threshold to judge186
whetherthefitdeviatedstronglyfromfirst-orderkineticsundernon-inhibitingconditions.187
• Extracted amounts from the solid phase: Since the trapped MPs are expected to be188
extractable from the solid phase along with the physically sorbed MPs, the extracted189
amounts should be higher under non-inhibiting than under inhibiting conditions. The time190
point with the maximal ratio between the extracted amounts from non-inhibiting and191
inhibiting conditions (after correction for differing total suspended solids concentrations192
underthetwoconditions)wasusedtoevaluatethiscriterion.Aratioof>1.9wasselectedas193
being indicative of significant accumulation in sludge under non-inhibiting conditions. This194
value was chosen because all compounds exhibited ratios of <1.9 in the recovery195
experiments(seeSIsectionS6.4andTableS6therein)anditisthereforeconsideredacrude196
estimateofthemaximaluncertaintyofthisratioduetouncertaintyinthesludgeextraction197
method.198
MPexperimentswereconductedinbioreactors(100and250mLSchottbottles)filledwithactivated199
sludge(50and100mL,respectively)sourcedfromthenitrificationbasinofafull-scaleSwissWWTP200
(moredetailsontheexperimentalset-uparegiveninChapterS2.2).Toachieveinhibitingconditions201
for protozoa, a digitonin solution (100mg/ml)20was added to selectedbioreactors to yield a final202
concentrationof600mg/L.After2hof incubation,experimentswerestartedbyspiking60µLofa203
MPsolution (50mg/L foreachMP) resulting ina startingconcentrationofabout60µg/L foreach204
MP.TomeasuretheliquidphaseconcentrationsoftheMPs,sampleswerewithdrawnfromtriplicate205
reactorsforeachconditionwithin35minutes(timezerosample)andatapproximately2h,4h,7h,206
12h,24h,30h,52h,and71hafterthestartoftheexperiment.TodeterminetheamountofMPsin207
thesolidphase,non-inhibitedandinhibitedactivatedsludgesampleswereextracted24at4h,24h,208
and 72 h. For this, filtered sludge samples from the reactorswere freeze-dried. After addition of209
internalstandards,thefollowingextractionprocedurewasrepeatedthreetimes.Extractionsolution210
(nanopure water:methanol:formic acid 200:200:1) was added, the mixture was vortexed,211
ultrasonicated (15 minutes at 50°C), and centrifuged (10 minutes, 4000 rpm, Megafuge 1.0 R,212
Heraeus).Thethusresultingsupernatantswerecombined,evaporatedtodrynessandreconstituted213
in nanopure water. Additional blank and recovery tests were conducted for the solid phase214
measurements. Additionally, sorption control experiments with autoclaved activated sludge and215
abioticcontrolexperimentswithautoclavedsludgefiltratewereconducted.MPswereanalysedby216
meansof reversed-phase liquid chromatography coupled to ahigh-resolutionquadrupoleOrbitrap217
massspectrometer(Qexactive,ThermoScientific)(adetaileddescriptionoftheanalyticalmethodis218
given in Chapter S3).7 Finally, another set of biotic reactors were run to measure specific219
9
experimentalconditions,includingpH,oxygenuptakerate,ammoniauptakerate,nitrateformation220
rate,andtotalsuspendedsolidsconcentration.221
222
ResultsandDiscussion223
Distributionofacridineorangeinactivatedsludge224
Figure2andFiguresS1-S7intheSIshowexamplesofthedistributionofacridineorange(AO)inthe225
activatedsludgeflocs. Inthesefigures,regionsemittinggreenorredlightarerenderedingreenor226
redcolor, respectively,whereasareasemittingbothgreenandred lightare rendered inyellow. In227
sludge samples without AO addition, no autofluorescence could be detected under the imaging228
conditionsused(FigureS8intheSI),confirmingthatanydetectedfluorescenceintreatedsamplesis229
associatedwiththepresenceofAO.230
In Figure 2, individual protozoa (identified as amoeboidsVahlkampfia) can be recognized and are231
clearlydistinguishablefromsludgeflocs(Figure2a).Theyshowedintensegreenandredfluorescence232
inclearlydelineatedcellularcompartmentsindicatingthatAOaccumulatedinthesestructures(note233
thatyellowareasinFigure2indicatesimultaneousemissionofbothgreenandredlight).Incontrast,234
sludgeflocsemittedashadedgreenishlightonly,mostlikelycausedbysorptionofAOtothesludge235
flocs or by binding of AO to bacterial DNA. Similar non-localized and less intense green light236
emissionscouldalsobeobservedinthecytosoloftheprotozoa.Totheextentthattheprotozoain237
Figure2andFigures S1-S7 couldbe identifiedbasedonvisual inspectionunder themicroscope, it238
was found that all amoeboids, some of the ciliates and none of the rotifers showed distinct239
fluorescentcompartments.Onlygreenfluorescencewasobservedwhensludgewasincubatedwith240
digitonin prior to adding AO (Figure S9 in the SI). Also, no protozoa could be recognized in these241
cases, confirming complete destruction of protozoa by digitonin.20 Together, these observations242
providestrongevidencethatAOwashighlyselectivelyaccumulatedinspecificcellularcompartments243
of certain groups of live protozoa in activated sludge. These observations not only confirmed the244
existenceofacidicvesicles inthoseprotozoa,butalsovisuallydemonstratedtheaccumulationofa245
specificamine-containingcompoundinthosevesicles.246
247
10
248
Figure 2: Confocal laser-scanningmicroscope images of activated sludge stainedwith acridine orange (AO).249
EmissionsfromAOmonomers,i.e.lowconcentrationsofAO,aredetectedatwavelengthsof480-560nmand250
showningreen.EmissionsfromAOstacks,i.e.highconcentrationsofAO,aredetectedatwavelengthsof590-251
660nmandshowninred.Yellowareas indicateemissionofbothgreenandred light. (a)Sludgeflocswitha252
protozoa(amoeboid,Vahlkampfia)attached;(b)Isolatedprotozoan(amoeboid,Vahlkampfia).253
254
b) a)
11
Fateofmicropollutantsundernon-inhibitingandinhibitingconditions255
Experimental parameters of the sludge communities in the bioreactor experiments (i.e., pH, total256
suspended solids concentration, oxygen uptake rate, ammonia uptake rate, and nitrate formation257
rate)undernon-inhibitingandinhibitingconditionsaregivenanddiscussedinSectionS6.1oftheSI.258
Briefly,observedtrendsinthetotalsuspendedsolidsconcentrationandtheoxygenuptakeratewere259
consistentwiththeabsenceofprotozoagrazingonbacteria inthebioreactorsrununder inhibiting260
conditions,whilenitrificationdidnotseemtobeaffectedbydigitonintreatment.261
ThemeasuredamountsofallinvestigatedMPsandtheirquantifiedTPsintheliquidandsolidphase262
ofactivatedsludgeincubatedunderbothinhibitingandnon-inhibitingconditionsaregiveninFigures263
S11-S30.Exemplaryresultsforfourcompoundsrepresentingdistinctlydifferentbehaviors(TRI,AZO,264
FEN,MIA)aregiveninFigure3.CalculatedrateconstantsandsorptioncoefficientsaregiveninTable265
2,TableS7,andTableS8.Accordingtotheresultsofthecontrolexperiments,sorptionandabiotic266
transformation were not affected by the addition of digitonin. The data also showed that abiotic267
transformationwas ofminor relevance and could be neglected. Results on the three criteria that268
wereassessedtocomparethebehavioroftheMPsundernon-inhibitingandinhibitingconditionsare269
presented in Table 2, namely concentration levels in liquid phase (as difference between mean270
concentrations), removal kinetics in the liquid phase (as R2 of the linear fit to the logarithmized271
concentrations), and extracted amounts from the solid phase (as maximal ratio of extracted272
amounts). Additionally, plots of the logarithmized concentrations against time, including linear fits273
andresidualplots,aregiveninFiguresS31-S50.274
Basedontheirbehaviorinthebioreactorexperiments,wecouldclassifytheMPsintothreegroups,275
wherebytwocompoundswerejudgedexceptions.ControlgroupI includedSUL,NAP,TRI,DET,and276
ALA,controlgroupIIincludedAZO,ISO,andCLT,andthetargetgroupincludedallamines(RAN,VEN,277
LID, TRA, LEV, MEX, FEN, CIT, PRO, andMIA) except for the two exceptions ATE and TIC. In the278
following,thesegroupswillbediscussedseparately.InFigure3,wepresentplotsofconcentrationas279
functionoftimeforatleastonerepresentativeofeachgroup.280
ControlgroupI.ThefivecompoundsofcontrolgroupIcouldnotbetrappedinacidicvesiclesdueto281
theirspeciationcharacteristics,whichareneutral-anionicforSUL,NAP,andTRI,andneutralforthe282
twocompoundsDETandALA.Therefore,onlybiotransformationandsorptionremainedaspossible283
removalprocesses,wherebythedatashowedthatthelatterwasofminorrelevance.Ascanbeseen284
in Figure 3a for TRI and in Table 2 for all five compounds, the deactivation of the protozoic285
community did not affect the removal of these compounds, since no significant difference in286
concentration-time courses was observed under non-inhibiting and inhibiting conditions, and287
removalfromtheliquidphasefollowedfirst-orderkineticsunderbothconditions.Thisindicatesthat288
12
protozoic biotransformation was not relevant for theseMPs and that the main removal process,289
namelybacterialbiotransformation,wasnotaffectedbytheadditionofdigitonin.290
Control group II. For the three fully neutral compoundsAZO, ISO, and CLT of control group II, ion291
trappingcouldalsobeexcludedasremovalprocess,andsorption,too,wasshowntobenegligible.292
Therefore, only bacterial and protozoic biotransformation remained as potentially relevant293
processes.Thismatchedtheobservationthattheirremovalfollowedfirst-orderkineticsunderboth294
conditions and that the extracted amounts from the solid phase were not significantly different295
betweenconditions.However,ascanbeseenfromtheconcentrationlevelsintheliquidphase(for296
AZO see Figure 3b; for ISO and CLT see Figures S17 and S18, respectively) biotransformationwas297
affectedbytheadditionofdigitonin.ThiswassupportedbytheconcentrationsofthetwoTPsAZOA298
and NISO, which were both formed in higher amounts under non-inhibiting conditions. The299
differenceinTPformationbetweenthetwoconditionswasmostpronouncedforAZOA,whichwas300
formedquantitatively fromAZOundernon-inhibiting conditions, yetwasnot formed in significant301
amounts under inhibiting conditions. Since it can be assumed that digitonin did not affect the302
bacterialactivity,20whichissupportedbyourmeasurementsoftheexperimentalparametersandthe303
resultsforcontrolgroupI,weconcludethatAZO,ISO,andCLTwereremovedfromtheliquidphase304
through biotransformation by protozoa. Interestingly, a common characteristic of all three305
compoundsisthattheypossessahydrolysablemoiety(i.e.,carboxylicacidesterandureagroups).At306
the same time, lysosomes, which are one type of acidic vesicles present in eukaryotic cells, are307
knowntoharbordifferentdegradativeenzymesthatbelongtotheacidhydrolasefamily.25Basedon308
ourresults,wethusspeculatethatAZO, ISO,andCLTarebiotransformedtoasignificantextentby309
hydrolasescontainedinprotozoiclysosomes.310
Target group. For all amine-containing MPs, except for ATE and TIC, which will be discussed311
separately,measuredamountsintheliquidphaseweresignificantlylowerundernon-inhibitingthan312
under inhibiting conditions,which indicates thatprotozoawere very important for the removal of313
amines from the liquid phase. Furthermore, the clear deviation from first-order kinetics and the314
higherextractedamountsfromthesolidphaseinthenon-inhibitedsludgecomparedtotheinhibited315
sludge(Table2)clearlypointedtowardsiontrappingbeingtheimportantprotozoicremovalprocess.316
MPs that were also removed under inhibiting conditions followed a first-order removal process317
(TableS8andFiguresS31-S50),indicatingthatbacterialbiotransformationwastherelevantremoval318
processforthemunderconditionswhereprotozoawereinhibited.319
320
13
Table 2: Sorption Coefficients, Removal Rate Constants, and Evaluation of Target and Control Compound321
Behavior(summarizedas“Levelofagreement”inthelastcolumn).322
Sorptioncoefficient,Kd,1 (non-inhibited)[L/kg]
Removalrateconstant,(inhibited)[1/day]
Maximaldifferencebetweenmeanliquid phaseconcentrations[timepoint]
R2 of thelinear fit tothelogarithmized liquidphaseconcentrations (non-inhibited)
Maximal ratiobetween theextractedamounts fromthe solid phase[timepoint]
Level ofagreementwith thethreecriteria toassesstargetbehavior
SUL -17(±21) 1.56(±0.03) 4.2[24h] 0.98 -
NAP -1(±11) 2.10(±0.15) 0.9[31h] 0.94 1.2[24h]
TRI -53(±16) 3.22(±0.19) 1.4[7h] 0.94 1.2[4h]
DET -14(±13) 1.17(±0.07) 0.5[7h] 0.90 1.2[24h]
ALA 45(±16) 1.91(±0.03) 7.2[12h] 0.99 0.9[4h]
AZO 19(±24) 0.03(±0.01) 42.4[52h] 0.99 0.8[4h] +
ISO 1(±18) 0.15(±0.01) 14.2[71h] 0.95 1.0[4h] +
CLT 6(±13) 0.36(±0.01) 22.4[52h] 0.88 0.9[4h] +
ATE -17(±20) 3.56(±0.19) 5.8[7h] 0.96 0.8[4h]
RAN 52(±19) 0.99(±0.05) 21.5[7h] 0.50 - ++
VEN 2(±14) 0.04(±0.01) 18.8[71h] 0.65 3.0[72h] +++
LID -5(±18) 0.08(±0.01) 26.1[31h] 0.84 6.8[24h] +++
TRA -2(±16) 0.05(±0.01) 18.1[31h] 0.63 4.0[24h] +++
LEV 227(±36) 0.23(±0.01) 18.4[31h] 0.83 3.1[24h] +++
MEX 190(±34) 0.48(±0.03) 23.0[7h] 0.70 2.7[4h] +++
FEN 36(±23) 0.62(±0.02) 23.0[7h] 0.63 7.2[72h] +++
CIT 199(±29) 0.62(±0.02) 10.7[7h] 0.75 2.9[72h] +++
PRO 231(±34) 0.58(±0.02) 16.5[7h] 0.76 1.4[72h] ++
MIA 570(±76) 0.53(±0.01) 11.8[7h] 0.77 3.9[72h] +++
TIC 5274(±906) 1.16(±0.05) 1.1[24h] 0.98 1.0[4h]
Data are given asmean and standard deviation. Sorption coefficients, Kd,1, calculated from the liquid phase323
concentrations in the abiotic and sorption control experiments as given in Equation S4 in the SI (for more324
details on calculation of sorption coefficients, including alternative methods and explanation of negative325
values, seesectionS6.5 in theSI).Removal rateconstantscalculated fromthe linear fit to the logarithmized326
14
data.Valuesjudgedindicativeofexpectedtargetcompoundbehaviorarehighlightedinbold(i.e.>8.7µg/Lfor327
the concentration difference in the liquid phase, <0.88 for R2, and >1.9 for themaximal ratio between the328
extractedamounts).329
330
331Figure3:Plotsofmeasuredamounts(innmol)asafunctionoftimefora)trinexapac-ethyl(controlgroupI),b)332
azoxystrobin(controlgroupII),c)fenfluramine,andd)mianserin(bothtargetgroup)undernon-inhibiting(left333
graph) and inhibiting (right graph) conditions. The following TPs are shown: b) azoxystrobin acid, c)334
fenfluramineN-desethyl,andd)mianserinN-oxideasTP1andmianserinformamideasTP2.Amountsinliquid335
and solid phase were calculated for a typical bioreactor with 100mL sludge and are shown asmeans and336
standard deviation of replicate measurements (n≥3) (If the error bar is not visible, it is smaller than the337
symbol).Totalamounts intheliquidphaseandparentaswellasTPamounts inthesolidphasearegivenfor338
thetimepointswherebothliquidandsolidphaseamountsweredetermined(4h,24h,72h).Fortheparent339
compounds, first-order fits are indicated as solid lines. TP amounts in the liquid phase are connected with340
dotted lines. TP amounts in the liquid phase are only shown if the amounts are higher than 1% of the341
theoreticalamountofparentspiked.342
343
Withinthetargetgroup,FEN,LEV,LID,MEX,TRAandVENbehaveveryconsistentlyasrepresented344
byFENinFigure3c.Ofthose,thebehaviorofVENandTRAismosteasilyinterpreted(seeFiguresS21345
andS23,respectively).Inbothcases,theamountsofTPsformedwereminorunderbothconditions346
andhardly any removalof theparent compoundswasobservedunder conditionswhereprotozoa347
wereinhibited.Itcanbeconcludedthatthesetwocompoundswerehardlybiotransformedatalland348
that the observed disappearance of the parent compounds from the liquid phase of the non-349
inhibited sludge was almost exclusively due to ion trapping. This is also supported by the larger350
amountsofparentcompoundsextractedfromthesolidphaseofthenon-inhibitedsludgerelativeto351
theinhibitedsludge(i.e.,ratiosof4.0at24handof3.0at72hforTRAandVEN,respectively),and352
0
5
10
15
20
25 a) Trinexapac-ethyl non-inhibiting
parent, liquid phase exponential fitTP1, liquid phaseTP2, liquid phasetotal, liquid phaseTPs, solid phaseparent, solid phase
inhibiting
0
5
10
15
20
25 b) Azoxystrobin non-inhibiting inhibiting
0
5
10
15
20
25 c) Fenfluramine non-inhibiting
0 10 20 30 40 50 60 70
inhibiting
0 10 20 30 40 50 60 70
0
5
10
15
20
25 d) Mianserin non-inhibiting
0 10 20 30 40 50 60 70
inhibiting
0 10 20 30 40 50 60 70Time [h]
Am
ount
[nm
ol]
15
thefactthattheparentcompoundsandTPsinthesolidandliquidphaseofthenon-inhibitedreactor353
summeduptowithin± 20%ofthetheoreticallyspikedamountatalltimepoints.354
Findings for LID, FEN, LEV and MEX are similar to those for VEN and TRA, yet more bacterial355
biotransformationwasobservedforthesecompoundsastheywerestillremovedininhibitedsludge356
and did so following first-order kinetics. While, for LID, consideration of the two quantified TPs,357
namelyNLIDandLINO,yieldedaclosedmassbalance(seeFigureS22),thiswasnotthecaseforFEN,358
LEVandMEX(seeFigures3c,S24,andS25,respectively).Forthelatterthreecompounds,thesumof359
all species in the bioreactors decreasedover time,whichwasmost likely due to the fact that the360
quantifiedTPswerenot stable,but transformed furtherduring thecourseof theexperiment.This361
alsoseemedtobethecaseforthemorestronglysorbingcompoundsCIT,PRO,andMIA.362
TP analysis additionally provided some evidence that biotransformation proceeded faster under363
inhibiting conditions than under non-inhibiting conditions for some of the compounds that364
underwentbacterialbiotransformation.ForFENandMIA,forinstance,theirrespectiveTPsNFENand365
MINO are formed in considerably larger amounts in the inhibited case (see Figures 3c and 3d,366
respectively).Thisseemsconsistentwithourhypothesisthatunderinhibitingconditionsmoreparent367
compound is available for biotransformation than under non-inhibiting conditions where a large368
fractionoftheparentistrappedinprotozoa.However,therewerealsocompounds,e.g.,LIDandCIT369
(seeFiguresS22andS27,respectively),forwhichformationofTPswaslessaffectedbytheinhibition370
oftheprotozoa.Finally,forRAN,forwhichtheamountinsludgecouldnotbequantifiedbutwhose371
behaviorwasconsistentwithiontrappingwithrespecttotheothertwocriteria,theformationofthe372
majorTPRASOwasevenfasterinthenon-inhibitedsludge.ThissuggeststhatinthecaseofRAN(see373
FigureS20), similarly to thecontrolcompounds incontrolgroup II,biotransformationwas tosome374
extentdirectlyaffectedbytheadditionofdigitonin,likelybecauseprotozoicbiotransformationwas375
alsorelevantforthisMP.376
For the strongly sorbing amines CIT, PRO,MIA (see Figures S27, S28, and 3d, respectively) and to377
someextentalsoforLEVandMEX,plotsofmeasuredamountsasafunctionoftimedeviatedfrom378
thoseof thepreviouslydiscussedamines.Theextractedamounts fromthesolidphaseof thenon-379
inhibitedand the inhibited sludgewerealmost the same.No significantdifferenceof themaximal380
ratiosofextractedamountswasobservedforPROatalltimepoints,andforCITandMIAatthefirst381
twotimepoints(i.e.,ratiosof1.4forCITandMIAat4h,and1.5forCITand1.7forMIAat24h).Yet,382
the data clearly show that the extracted amounts of CIT andMIA from the non-inhibited sludge383
remainedfairlyconstantoverthecourseoftheexperiment,whilethoseextractedfromtheinhibited384
sludgedecreasedwithtime(ratiosof2.9forCITand3.9forMIAat72h).Thissuggeststhatunder385
inhibitingconditionsmostofthecompoundsextractedfromthesolidphasewerereversiblysorbed386
and consequently equilibrated with the dissolved fraction, which was available for387
16
biotransformation. In contrast, under non-inhibiting conditions,most of the extracted compounds388
camefromthetrappedcompoundpool,whichwasnotavailableforbiotransformation.ForMIA,this389
explanation is consistent with the observed increased formation of its TP MINO under inhibiting390
conditions. Overall, these observations suggest that for the strongly sorbing amines, ion trapping,391
reversiblesorption,andbiotransformationwerehappeningveryreadilyandonsimilartimescales.392
ThetwomajorexceptionswithrespecttothetypicalpatternsobservedforaminesareATEandTIC393
(seeFiguresS19undS30,respectively).WhileATEshowedverylittlesorptiontosludge,TICwasthe394
most strongly sorbing amine studied here. Neither of them showed a clear difference in the395
concentration-timeplots betweennon-inhibiting and inhibiting conditions (see Table 2), indicating396
that iontrappingdidnotoccurtoanyrelevantextent.Whilethereasonsforthesefindingsremain397
largelyelusive,itisnoteworthythatthetwocompoundsexhibitedthefastestbiotransformationrate398
constantsunder inhibitingconditionsofallaminesstudied(Table2).ForATE, itsTPATAC,which is399
known to be formed through enzyme-catalyzed hydrolysis,26 was formed nearly quantitatively,400
confirming ATE removal to be due to biotransformation. Based on these findings, it could be401
hypothesized thatATEandTICwere transformed so readily in the cells’ cytosol thatno significant402
trappingoccurred.403
Allinall,thedatafromimagingexperimentswithacridineorangeaswellasfromexperimentswith404
amine-containingMPs in activated sludge with and without inhibition of protozoa provide strong405
evidence that ion trapping of amine-containing MPs occurs in the acidic vesicles of protozoic406
eukaryotes present in activated sludge. Furthermore, the experiments with the control MPs also407
indicatedthatprotozoicbiotransformationisrelevantforsomeMPs,suchasAZO,ISO,CLT,andRAN,408
whereasbacterialiontrappingseemedtobeofminorimportanceintheactivatedsludgeusedinour409
experiments.410
411
Additionalexperimentalevidenceforiontrapping412
Beyond theexperimentsdescribed indetail here, results fromourprevious researchonamines in413
activated sludge provide evidence for other characteristics of the ion trapping process. First, the414
extentof trappinghasbeendescribed to increasewithelevatedextracellularpH levels.12Wehave415
observed and described an increase in removal rate constants of amine-containing MPs with416
increasing pH in previous experiments conducted at pH 6, 7 and 8.7 While the observation was417
correctly interpretedasbeingdue to thedifference inmembranepermeabilityof thecationicand418
neutralspeciesoftheamines,wemostlikelyincorrectlyattributedtheobservedlossoftheamines419
to biotransformation only. Based on the findings presented here and the fact that, also in the420
previousstudy,theremovalkineticsdidnotalwaysfollowfirst-orderkinetics(e.g.,forPAR,MIA,ORP421
17
and PYR in 7), we now assume that at least part of the observed, pH-dependent removal in the422
previousstudywasalsoduetoiontrapping.423
Second,itisknownthatthevitalityofthecellsinfluencestheirtrappingcapacity.9,27,28Thisisrelated424
to the fact that themaintenanceof the lowpH level in theacidic vesicles is anactivemechanism425
consumingenergy.Instressedcells,energysupplyislimited,whichreducesthetrappingcapacityof426
theacidicvesicles.Inourpreviouswork,wehaveobserveddecreaseddisappearanceofthestudied427
amines from the liquid phase under conditions where the reactors were stirred with magnetic428
stirrers7ascomparedtowhensludgesuspensionwasmaintainedbyshaking10only.Stirringofthe429
sludgeaffectstheintactnessofthesludgeflocsbyexertingmechanicalstress.Thismightnegatively430
affecttheprotozoa,whichneedintactsludgeflocstoattachto.However,sinceourdataallowsthis431
comparisonforamine-containingMPsonly,itremainsunclearwhicheffectstirringorshakinghason432
otherremovalprocessessuchasbacterialbiotransformation.433
Finally,ithasbeenshowninmammaliansystemsthatthetrappingcapacityoftheacidicvesiclescan434
be saturated.29 Thus, ifmixtures of several positively ionizable compounds are present, their total435
concentrationmightexceedasaturationthreshold,leadingtolesstrappingforeachofthemthanif436
presentindividually.Inourpreviousstudy10,amineswerespikedindividuallyintobioreactorsorina437
mixture of ten. As can be seen from the measured concentrations given in the supporting438
informationofthatpaper,concentrationlevelsintheliquidphasewerehigherwhenthemixturewas439
spikedthanwhenthecompoundswerespikedindividually(e.g.,differencesaremostpronouncefor440
VEN, LID and PHE). This confirms that the saturation of the acidic vesicles also occurs within the441
protozoiccellsoftheactivatedsludgecommunity.442
443
Implications444
Taken together, the results of this study and previous experimental observations provide strong445
evidencethationtrappingisanadditional,importantremovalmechanisminbatchexperimentswith446
activated sludge for a highly prevalent class of MPs, i.e., aliphatic amines, that has so far gone447
unnoticed.Asaconsequence,ithasoftenbeenmisinterpretedasbiotransformationbyresearchers448
studying the fate of amines in activated sludge, including ourselves.7, 10, 30, 31 Here, we provide a449
mechanisticunderstandingof the ion trappingprocess,whichweobserved tooccur fora rangeof450
aliphaticamineswithpKavaluesbetween7to10andlipophilicitiesrangingbetweenlogPof0.4and451
4.2.Wealsobrieflydiscusstheinfluenceofdifferentexperimentalfactorsontheobservationofion452
trapping during batch experiments,which should enable recognition and correct interpretation of453
thisremovalprocessinfuturestudies.454
Beyonddemonstratinganovelremovalmechanisminbatchexperimentswithactivatedsludge,our455
observationstriggeranumberof follow-upquestionsthatarerelevantfordifferentscientific fields456
18
andpractical applications. First, how relevant is ion trappingof amine-containingMPs in full-scale457
systems? Since we worked with sludge sourced from a full-scale WWTP and still observed ion458
trapping,weconcluded that thecapacityof theacidicvesicles for takingupadditionalcompounds459
was not exhausted under the conditions of the experiments. However, more quantitative460
investigationson the typesand relativeamountsofprotozoicbiomass involved inaccumulationof461
aminesinactivatedsludgewouldbeneededtoestimatethepotentialmagnitudeoftheeffectinfull-462
scale systems. Second and potentially evenmore importantly, one could ask if not only aliphatic463
amines, but also ammonium itself accumulates in the acidic vesicles of protozoa. This thought is464
backed up by the fact that NH4Cl is used in cellular biology to alkalinize acidic vesicles.8, 32-34465
Additionally, own preliminary experiments with different sludge communities indeed pointed466
towards a short-term increase in ammonium concentrations upon destruction of eukaryotic cells467
throughdigitoninaddition(datanotshown).Storageofammoniuminprotozoa inactivatedsludge468
could potentially have far-reaching implications for our current view of the nitrogen cycle during469
wastewater treatment and might explain some of the irregular behaviors (i.e., sudden onsets of470
incompletenitrogenremoval)observedinfull-scaletreatmentplants.Additionally,sinceammonium471
concentrations during activated sludge treatment are several orders of magnitude higher than472
micropollutant concentrations, trapping of ammonium would potentially outcompete trapping of473
amine-containing compounds in full-scale systems. Third, a specific type of acidic vesicles, the so-474
called acidocalcisomes, have also been described to occur in bacteria, in particular in phosphate-475
accumulatingbacteria.35,36Thisraisesthequestionwhetherbacterialiontrappingalsocontributesto476
theextentof trappingofamine-containingMPsand/orammonium inactivatedsludge fromplants477
with enhanced biological phosphorus removal. Fourth, amine-containing compounds and/or478
ammonium trapped in acidic vesiclesmight be re-released from sludge upon cell disruption, e.g.,479
during anaerobic sludge digestion. Since the majority of micropollutants is expected to be480
recalcitrant to biodegradation under anaerobic conditions,37 we expect amine-containing481
micropollutants thus released to be transported out of the WWTP together with the biosolids.482
Finally, ion trapping may be a relevant process not only in microbial communities, but also in483
eukaryoticorganismsrelevant inenvironmentalsciences ingeneral,suchastestorganismsused in484
ecotoxicological studies. Taking this into consideration would require re-interpretation of the485
toxicokineticbehaviorofpositivelyionizablecompoundsinsuchsystems.486
487
Acknowledgments488
TheauthorsthankthestafffromtheWWTPNeugut,Dübendorf,Switzerland,forprovisionofsludge489
and help with sludge sampling. Martin Ackermann, Eawag and ETH Zürich, Switzerland, is490
acknowledged for letting us use the Laser Scanning Confocal Microscope and Joachim Hehl from491
19
ScopeM, ETH Zürich, Switzerland, for providing support with the instrument. We further greatly492
thank André Wullschleger from the WWTP Werdhölzli, Zürich, Switzerland for annotation of the493
protozoa. Financial support for this project has been provided by the European Research Council494
under the European Union's Seventh Framework Programme (ERC grant agreement no. 614768,495
PROduCTS)andbytheSwissNationalScienceFoundation(projectno.200021_134677).496
SupportingInformation497
Details on materials and methods, including MP selection, batch experiments, measurement498
methods, and data analysis, and details on results, including microscope images, experimental499
parameters,MPamounts in liquid and solidphasewithevaluationanddiscussion. Thismaterial is500
availablefreeofchargevietheInternetathttp://pubs.acs.org.501
502
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