Intrinsically Green Iron Oxide Nanoparticles? From Synthesis via (Eco-)Toxicology to ScenarioModellingJuliane Filser*a,b,c, Darius Arndta,b,d, Jonas Baumanna,b,c, Mark Gepperta,b,e,Stephan Hackmanna,b,c, Eva M. Luthera,b,e, Christian Padea,f, Katrin Prenzela,g, Henning Wiggera,f, Jürgen Arninga,b,c,h, Michaela C. Hohnholta,b,e, Jan Kösera,b,h, Andrea Kücka,b,h, Elena Lesnikova,b,c, Jennifer Neumanna,b,h, Simon Schütrumpfa,b,h, Jürgen Warrelmann*a,b,c, Marcus Bäumera,b,d, Ralf Dringena,b,e, Arnim von Gleich,a,f Petra Swidereka,g and Jorg Thöminga,b,h

Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XXDOI: 10.1039/b000000x

Iron oxide nanoparticles (IONP) are currently being studied asgreen magnet resonance imaging (MRI) contrast agents. They arealso used in huge quantities for environmental remediation andwater treatment purposes, although very little is known on theconsequences of such applications for organisms and ecosystems.In order to address these questions, we synthesisedpolyvinylpyrrolidone-coated IONP, characterised the particledispersion in various media and investigated the consequencesof an IONP exposure using an array of biochemical andbiological assays. Several theoretical approaches complementedthe measurements. In aqueous dispersion IONP had an averagehydrodynamic diameter of 25 nm and were stable over six days inmost test media, which could also be predicted by stabilitymodelling. The particles were tested in concentrations of up to100 mg Fe/L. The activity of the enzymes glutathione reductaseand acetylcholine esterase was not affected, nor wereproliferation, morphology or vitality of mammalian OLN-93 cellsalthough exposure of the cells to 100 mg Fe/L increased thecellular iron content substantially. Only at thisconcentration, acute toxicity tests with the freshwater fleaDaphnia magna revealed slightly, yet insignificantly increasedmortality. Two fundamentally different bacterial assays,anaerobic activated sludge bacteria inhibition and a modifiedsediment contact test with Arthrobacter globiformis, both renderedresults contrary to the other assays: at the lowest testconcentration (1 mg Fe/L), IONP caused a pronounced inhibitionwhereas higher concentrations were not effective or evenstimulating. Preliminary and prospective risk assessment wasexemplified by comparing the application of IONP withgadolinium-based nanoparticles as MRI contrast agents.Predicted environmental concentrations were modelled in twodifferent scenarios, showing that IONP could reduce theenvironmental exposure of toxic Gd-based particles by more than50%. Application of the Swiss “Precautionary Matrix forSynthetic Nanomaterials” rendered a low precautionary need forusing our IONP as MRI agents and a higher one when using themfor remediation or water treatment. Since IONP and(considerably more reactive) zerovalent iron nanoparticles arebeing used in huge quantities for environmental remediationpurposes, it has to be ascertained that these particles pose norisk to either human health or to the environment.

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____________________________________________________a University of Bremen, D-28334 Bremen, Germanyb UFT Centre for Environmental Research and Sustainable Technologyc Department of General and Theoretical Ecology. Fax: +49 421 2187654; Tel: +49 421 218 63470; E-mail: filser@uni-bremen.ded Institute for Applied and Physical Chemistry. Fax: Fax: +49 421 21863188; Tel: +49 421 218 63170; E-mail: mbaeumer@uni-bremen.de e Centre for Biomolecular Interactions Bremen. Fax: +49 421 21863230; Tel: +49 421 218 63244; E-mail: ralf.dringen@uni-bremen.def Department of Technological Design and Development. Fax: +49 421 2187503; Tel: +49 421 218 64880; E-mail: gleich@uni-bremen.deg Institute for Applied and Physical Chemistry. Fax: +49 421 2163188; Tel: +49 421 218 63200; E-mail: swiderek@uni-bremen.deh Department of Sustainable Chemical Engineering. Fax: +49 421 2188297; Tel: +49 421 218 63300; E-mail: thoeming@uni-bremen.de

† Electronic Supplementary Information (ESI) available: Full experimental methods, additional results (Tables S1-S6, Figs S1) andextended background literature. See DOI: 10.1039/b000000x/

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IntroductionLarge amounts of various nanomaterials havealready been marketed due to new or enhancedqualities that allow for new functionalities1;2;3, but there is still insufficientknowledge regarding the environmental,health, and safety risks that thesematerials might involve 4;5;6;7. Additionally,well established conventional methods ofrisk assessment may not be applicable tonanomaterials 8;9;10;11;12. Hence, there is aclear need for a preliminary and prospectiverisk assessment of nanomaterials 13.Our study deals with iron nanoparticles

which can be roughly grouped in nanoscaledzerovalent iron (nZVI) and iron oxidenanoparticles (IONP). Possible applicationsof IONP are, e.g., data storage,(bio)remediation, or biomedical applications14-16. Magnetite (Fe3O4) is used as a contrastagent for magnet resonance imaging (MRI) 17.IONP could replace the currently dominatinggadolinium (Gd) based contrast agents 18. Gdis toxic in free form 19 and is thereforechelated in diverse complexes for MRIapplications. For MRI it has to be processedinto a pure form under high effort with acorresponding ecological footprint.Given the low cost of the technology and

the huge amount of contaminated sites, thequantitatively most important application ofiron nanoparticles is environmentalremediation of ground and surface water,sediments and soils. Here, overwhelmingquantities of particles (mostly nZVI) aredirectly applied to the environment 20;21;22.The preparation of IONP which are stable

in different environments and media is stilla big challenge in colloidal chemistry. Manydifferent approaches were established in thepast to create and functionalize suchparticles in different synthetic ways 23,24. Amajor route is the co-precipitation method25,26 where the particles are functionalisedin situ 27 or in a subsequent step with aprotecting ligand shell 28. However, thismethod has the disadvantage that theparticles are usually not uniform in size.Another method, the thermal decomposition ofan organic iron precursor, leads to uniformwater insoluble particles 29,30 and wasapplied here.Beside some allergic reactions to

polysaccharide coated iron oxide contrastagents 31-33, toxicological data raise littleconcern for administering IONP to humans12,34-37. Some studies did find toxic effectsof IONP in relevant concentrations (e. g.Ying & Hwang, 2010 38). However, due tomissing or insufficient characterisation ofthe IONP, it often remains unclear whether

or not the measured toxicity was caused bythe IONP themselves or by impurities of orresidues in the (commercial) IONPsuspensions used 39. The degradation of ironoxide in the human body is well understood18. Besides direct medical administration12 ,to our knowledge studies investigating the(potential) exposure of humans to IONP havenot been published yet. This might be due tothe fact that exposure assessment ormodelling for nanoparticles still remains amethodological challenge 40.Very little is known about the

environmental toxicity of IONP 41-44, althoughsome more studies exist on nZVI. Since nZVIquickly oxidise unless kept under strictlyanaerobic conditions, the results of thesestudies should to some extent also apply toIONP. nZVI had little influence on daphnidswhereas negative effects were found formice, fish and bacteria 45, especially underanaerobic conditions 46. IONP instigateddevelopmental toxicity in zebrafish atconcentrations ≥ 10 mg/L44.The risk of substances that come into

contact with humans and the environment inlarge quantities has to be scrutinizedthoroughly. The UFT Research Centre forEnvironmental Research and Technology haslong-term experience in interdisciplinaryhazard assessment of novel substances,particularly of ionic liquids 47,48. Ourapproach, consisting of an(eco-)toxicological test battery on thegrounds of analytical skills, structure-activity relations of chemicals (SAR) andprofound ecological knowledge, has beencontinuously widened over the past ten years49,50Our test systems cover both human andenvironmental toxicology, involving numeroustests for various compartments and trophiclevels. They include simple enzymeinhibition assays, tests with cell cultures,single-species tests and communities, thusallowing to select appropriate subsets oftest systems for almost any exposuresituation conceivable. Recently, we havefocused on engineered nanoparticles 51,52

This study is to our knowledge the firstto cover manifold aspects of IONP, fromsynthesis and characterisation to riskassessment on the grounds of a substantialbody of test systems and theoreticalapproaches. We chose a set of testsinvolving various biochemical andphysiological assays, a cell culture, afreshwater crustacean and both aerobic andanaerobic bacteria. In addition, we usedvarious theoretical approaches for stabilitymodelling and for a preliminary andprospective environmental risk assessment.

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ExperimentalHere a general overview of methods used inthis study is given. Materials, moredetailed background and descriptions ofsingle methods can be found in theElectronic Supplementary Information (ESI).Particle synthesis and characterisationSynthesis of PVP-coated magnetite (Fe3O4)IONP. To create uniform and colloidal stablemagnetite IONP with a core size of 5.7 ± 0.5nm in a simple one pot reaction, we chosethe thermal decomposition of iron (III)acetylacetonate in a highly boiling polyolliquid 53. Additionally, we addedpolyvinylpyrrolidone 58k MW (PVP) duringthe reaction to achieve a better resistanceof the IONP against agglomeration andprecipitation, processes that especiallyoccur when the particles are used in highionic strength or protein bearingenvironments. 6.25 mM PVP 58k were dissolvedin 25 mL diethylenglycol (DEG) and heated upunder stirring to 80 °C. After 30 minutes1.25 mM iron (III) acetylacetonate wereadded and the solution was stirred foranother 30 minutes. The reaction medium wasthen heated up to 220 °C where it was keptfor 2 h. To separate the particles from DEG,30 mL acetone were added per mL reactionmixture and the precipitate was centrifuged.The powder that is composed of 75 wt% PVPand 25 wt% iron oxide was dried under airand re-dissolved in water. These PVP-coatedIONP were used in all tests and are, forconvenience, referred to as IONP in thefollowing. If not mentioned otherwise, allconcentrations are given in mg Fe/L.Preparation of IONP in physiological media.A stock solution of IONP was prepared with aconcentration of 1 g Fe/L in Milli-Q water.Before mixing with the pure media to a finalconcentration of 100 mg Fe/L, the stocksolution and the used media (see in vivo and invitro testing and Table S1) were sterilisedusing Millipore 0.2 µm filters. The durationof the settle time for the different mediawas predetermined by the environmental andbiological experiments and set to six daysfor water, Elendt M7 and DMEM-FCS and to twodays for DSM, AS, AchE and GR media.Total iron and free iron ion concentration.To control the total iron concentration ofthe stock solution and its mixtures withmedia, a modified iron assay was employed54. For this purpose, 100 µL of the PVP-coated IONP stock solution were diluted with100 µL NaOH (50 mM) and digested in 100 µLreleasing reagent (2.25% KMnO4/0.7 mM HCl)at 60 °C for 2 h. After digestion, 30 µLdetection reagent (aqueous solution of 2.5 M

ammonium acetate, 1 M ascorbic acid, 6.5 mMferrozine and 6.55 mM neocuproine) wereadded. The absorbance of the resultingferrous iron-ferrozine complex was measuredat 540 nm to quantify the iron content(Tecan Sunrise, Grödig, Austria). The totaliron concentration of theacetylcholinesterase (AchE) and glutathionereductase (GR) media was measured withAtomic Absorption Spectroscopy (AAS). An AAS5FL (Carl Zeiss AG, Oberkochen, Germany) wasoperated with an acetylene/nitrous oxideflame after centrifugation and diluting thesupernatant media with water to a finalconcentration of 10 mg/L.To estimate the free iron concentration,

the abovementioned ferrozine assay wasmodified. 30 µL detection reagent werediluted with 200 µL double distilled H2O andadded to the samples without any digestion.The total iron content was measured at 540nm immediately after addition of ferrozine.The results are quoted as percentage oftotal iron content.Particle properties. The stability of thePVP-coated IONP against forced sedimentationin different media was checked bycentrifugation of the samples at 1000 g(Minispin, Eppendorf, Germany) anddetermining the remaining concentration ofparticulate iron and dissolved iron ions inthe supernatant (see above). Particlediameter distribution (dynamic lightscattering, DLS) and zetapotential weredetermined using a Beckman-CoulterDelsaNanoC (Beckman Coulter GmbH, Krefeld,Germany). The data were acquired three timesfor three different samples. For comparison,we measured the pure medium (Blank) withoutany nanoparticles as well. In addition, weperformed a long-term stability test for oneyear in pure water.Biological and biochemical assaysDuring the course of all tests, we verifiedthe IONP concentration using the iron assaywith ferrozine (see above). Additionally,the nanoparticles were characterised by DLSas well as zetapotential measurements. The following assays were chosen for

screening the acute hazard potential of theparticles. Selection criteria were moleculartargets, cell metabolism, trophic position,importance for ecosystem functioning andrelevance for chemicals regulation. Alltests were carried out in at least threedifferent concentrations of IONP and acontrol and independently replicated threetimes if not mentioned otherwise. The IONPconcentrations ranged from 0.1 to 100 mgFe/L (different dimensions for microbialassays referring to solids).

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In vitro testingEnzymes: Glutathione reductase (GR) is acytosolic flavine-dependent enzyme thatcarries flavin adenine dinucleotide (FAD+)as prosthetic group near its active site. GRis a key enzyme in maintaining the highglutathione GSH/GSSG ratio within cells bybinding GSSG as natural substrate andreducing it to two molecules of GSH. Theelectrons needed for this reaction step areprovided by the cofactor nicotinamideadenine dinucleotide phosphate (NADPH). The“soft” Lewis-basic thiolate anion at theactive site of the enzyme renders GRextremely vulnerable for an inactivation bythe reaction with electrophilic, “soft”Lewis-acidic substances such as IONP. Theassay was performed according to 55. Forvalidation of the test, 5-chloro-methylisothiazol-3-on was used as positivecontrol.The enzyme acetylcholinesterase (AchE)

catalyses the rapid degradation of theneurotransmitter acetylcholine in thesynaptic cleft – one of the key mechanismsin neurotransmission in nearly all higherorganisms. An inhibition of AchE leads tovarious adverse effects in neuronalprocesses. The activity pattern of thisenzyme in different biological matrices andtissues is used as an established biomarkere.g. to monitor the pesticide burden in non-target species. The inhibition of the AchEwas measured using a colorimetric assay,which is described in detail in 56 Forvalidation of the test, carbendazim was usedas positive control. Neither here nor in theGR assay any interference of thenanoparticles with the assay set-up wasobserved.OLN-93 cells. The mammalian cell line OLN-93is a model for oligodendroglial brain cells57 These cells proliferate in serum-containing culture medium and accumulatediron from various low molecular weight ironsources 58. In addition, OLN-93 cells havebeen shown to take up various types ofnanoparticles, including citrate- ordimercaptosuccinate-coated iron oxidenanoparticles 59;58,60.OLN-93 cells (passage numbers between 37

and 39) were cultured as describedpreviously 61. Three independent experimentswere performed on three different passagesof cells. Protein content and cellularlactate dehydrogenase (LDH) activity of theOLN-93 cells were quantified as indicatorsof cell number and viability as recentlydescribed 61 The cellular iron content was quantified

using the ferrozine method describedpreviously. The colorimetric Tietze assay

was used to quantify the cellular contentsof GSx (GSx = amount of GSH plus twice theamount of GSSG) and GSSG as describedearlier 62.To test for the membrane integrity, the

membrane impermeable dye propidium iodide(PI) with counterstaining of the cell nucleiby H33342 was applied. Intracellularreactive oxygen species (ROS) were detectedby Rhodamine 123 staining 61

. The absence of any interference of thePVP-coated IONP with the colorimetric assaysystems was confirmed by standard additionof the particles to standard amounts ofprotein, GSx or lactate and by addition ofparticles to the cell lysates to determineLDH activity (data not shown). A quenchingby accumulated PVP-coated IONPs of PI canalso be excluded (data not shown), since aco-incubation of OLN-93 cells with PVP-IONPsplus AgNO3 did not lower the number of PI-positive cells compared to the AgNO3condition that severely compromises membraneintegrity (63). Also the rhodamine 123fluorescence observed after treatment ofOLN-93 cells with ferric ammonium citrateand H2O2 was not lowered in PVP-IONP-exposedcells (data not shown), demonstrating thatthe NPs do not quench ROS-induced rhodamin123 fluorescence.In vivo testingThree slightly modified standard assays forchemicals regulation were selected torepresent higher organisms andmicroorganisms. For the latter, we chose asingle-species test with aerobic bacteriaand an anaerobic community assay.Daphnia acute toxicity. The acuteimmobilisation test with Daphnia magna wasperformed according to the OECD-guideline202 64 with some adaptations‡. The number ofanimals per concentration was reduced from20 to 10, the overall amount of liquid wasreduced from 40 to 20 mL per concentration,and the number of replicates was increasedfrom 4 to 10. The test was performed inElendt M7 medium in 24-well cell culturingplates. In each well a fresh born neonate(<24 h) was exposed in 2 mL of the testsolutions with 10 replicates and 5 repeatsof the test. The test duration was prolongedto 96 h (48 h in the standard design). After24, 48, 72, and 96 h the immobilization ofthe daphnids was recorded by visualinspection. The solutions were not renewedduring the 96 h exposure and the daphnidswere not fed. Tests were only counted validwith an immobilization ≤ 10% in the controlafter 96 h. The daphnids were exposed toIONP concentrations of 0.1, 1.0, 10 and 100mg Fe/L.

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Contact test with Arthrobacter globiformis. Theaerobic, chemo-heterotrophic and non-pathogenic soil bacterium Arthrobacterglobiformis is one of the common bacteria innatural soils and sediments 65. Exposure totoxic substances in the aquatic orterrestrial environment is highly likely,e.g. when particles used for MRI purposeshave been excreted and pass through thewastewater treatment. Standing at the bottomof the nutrition chain, A. globiformis is animportant food source for organisms onhigher trophic levels 66,. The test systemapplied here is based on a modification ofthe German Standard Sediment Contact Testwith Arthrobacter globiformis (DIN 38412 L48(2002) 67,68, measuring dehydrogenaseactivity. The redox process catalysed by theenzyme dehydrogenase in vital bacterialcells is measured by chemical reduction ofthe blue dye resazurin to the pink productresorufin. Resazurin can be measuredspectrophotometrically (DIN 38412 L48) 67.Sewage sludge inhibition. Since themicrobial community of wastewater treatmentplants represents the first target forcontaminants from waste water streams anegative influence on its activity can leadto severe ecological consequences. To assessa realistic worst-case scenario theactivated sludge samples were taken from anindustrial waste water treatment plant.The assay was modified from OECD test

guideline No 224 (2007) 69 using anaerobicconditions, which are applied fordenitrifying biodegradation tests. Theinhibition was followed via the cumulativegas production of the bacteria over time,since metabolically active bacteria produceN2 and CO2 under anaerobic conditions. Theassay was performed with IONP concentrationsin a range between 0.01 and 1.5 mg Fe/mg TS(total solids).Data presentation and statistical analyses If not stated otherwise, data are shown asmeans ± standard deviations of threeindependent replicates. Differences betweendata sets were analysed by the t-test (twosets) or by ANOVA (groups of data) followedby the Bonferroni post hoc test using theopen source “R” software for statisticalcomputing or Graph Pad (STATCON,Witzenhausen, Germany).Modelling and scenariosColloidal stability of nanoparticles. It ispossible to simulate the colloidal stabilityof nanoparticles as a function of pH valueand ionic strength 70,71, thus reducing thenumber of time-consuming stabilityexperiments. We determined the theoretical

colloidal stability of our IONP in differentaqueous media using the Dejaguin-Landau-Verwey-Overbeek (DLVO) theory. Generally,there are two different types of DLVOtheory, the classic and the extended model.According to classic DLVO theory, thecolloidal stability of particles isdetermined by the sum of the repulsive netelectrostatic double layer interactionenergy of the particles (by definitionpositive values) and the van der Waalsattractive energy (negative values), e.g. anegative total interaction potential leadsto instability 72

In case of polymer covered IONP,additional forces such as magneticattraction and steric repulsion must beconsidered. With these additional terms theDLVO theory needs to be extended. The stericrepulsive potential consists of osmoticrepulsion and elastic steric repulsion. Thelocal osmotic pressure increases due to theoverlapped polymer layers of two particles.A rise of an elastic repulsion is induced bya compression of the adsorbed layers belowthe thickness of the unperturbed layer,which is accompanied by a loss of entropy70. Here we followed the calculation methodby Lim et al. 73.

Risk assessment. The main objective ofexposure assessment is to determine thepredicted environmental concentration (PEC)of a substance Together with the predictedno effect concentration (PNEC) it definesthe risk ratio PEC/PNEC, which is being usedfor prioritisation of further research andregulation. The assessment here used amethod for PEC estimation in surface waters,74. Based on available literature data onproduction, use and expected marketdevelopment in Germany until 2020, best-caseand business-as-usual scenarios weremodelled both for Gd and IONP based MRIcontrast agents.We applied one existing tool to

prospectively and preliminarily assessedpotential risks of nanomaterials, the Swiss“Precautionary Matrix for SyntheticNanomaterials” 75. It is based on a set ofquestionnaires, which assign a score(usually between 1 and 9) to each answer.The scores are processed in a formula, andthe resulting value renders the“precautionary need” for “workers ingeneral” (WG) and “workers in the worstcase” (WWC) as well as for “consumers” (C)and the “environment” (E). This value canrange between 0 (“no nanomaterial / norelevance“) and more than 7,000 (“extremelyhigh precautionary need”). There are fourcategories of questions:1 Nano relevance, e.g. particle size

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2 Specific framework conditions, e.g. knownorigin of the materials, consumers etc.

3 Potential effects, specifically knownstability, reactivity and damage

4 Potential exposure, e.g. annualproduction volume, medium in which theparticles are used etc.

For details, see Tables S2a and S2b as wellas 75.

Results and discussionParticle characterisation and stabilityFig. 1 and Table S3 show characteristics ofthe particle dispersions in a range ofdifferent media. Over the course of sixdays, the particles in stock solution (1 gFe/L) on average had a diameter of about 25nm and a zetapotential of 0.4 mV at pH 5.6.When diluted in water to 100 mg Fe/L, exceptfor the pH no significant change occurredover six days and even up to one year (datanot shown). As compared to pure water(Blank) we found no influence of the neutralPVP-coated IONP on the zetapotential.When the nanoparticles were suspended in

Elendt M7 with a concentration of 100 mg/L,we observed an increase of the particle sizewithin 6 days with the mean diameter raisingfrom 46 nm at day 0 to 232 nm at day 6 (Fig.1a). This can be attributed to the highconcentrations of Ca2+ and Mg2+ of thismedium (Table S1), leading to a higheragglomeration tendency of the PVP-coatedIONP. These ions are known to lead to strongagglomeration of colloids 76;77;78. Thisbehavior was not observed for suspensions ofIONP in DMEM-FCS medium with similarconcentrations of Ca2+ and Mg2+, which couldbe attributed to the stabilising effect ofthe proteins in the medium. 79;80.The DMEM-FCS medium contains protein,

complicating the interpretation of theresults. Even in the pure medium“nanoparticles” in a range of 28 nm weremeasured (Fig. 1a), possibly resulting fromthe protein itself. Therefore, it was notpossible to distinguish between the proteinand the PVP-coated IONP in these samples.Nevertheless, the results for the IONPdiluted with DMEM-FCS show that they did notundergo any significant agglomerationprocess: the hydrodynamic diameter increasedslightly to just 35 nm after 6 days, whichmight be attributed to the attachment of theprotein to the particles (Fig. 1a).Concerning the PVP-coated IONP in AchE, AS

, GR and DSM medium, only the AS sampleshowed a slight increase of the particlediameter from 25 nm at day 0 to 36 nm at day1 (Fig.1b). This could be attributed to the

small change of the zetapotential of IONP inmedium AS. For the other media there were nosignificant differences of the zetapotentialof the nanoparticle containing samplescompared to blank measurement of puremedium, as in all other cases (Fig. 1b).The measurements of iron content were in

good agreement with the nominalconcentrations, and with one exception notmore than 2% of the total iron was found insolution (Table 1), indicating no evidencefor dissolution or agglomeration of theparticles. Only in DSM we found a free ironconcentration up to 9% after 2 days. Wesuppose that the more reductive conditionsin this medium led to some particledissolution, resulting in free iron ions. Stability assessment. To assess thestability of the PVP-coated IONP, theirtotal interaction energy was modeled formedia with different ionic strengths. Thehydrodynamic diameter in water determined byDLS and the core diameter determined by TEMwere considered as size properties ofprimary IONP. In order to estimate thetendency of these primary IONP toagglomerate, the contributing interactionenergies were then calculated using the datafor ionic strength and zeta potential in thedifferent media (listed in Table S1 and S3).Since the zeta potential of the particlesare all close to zero there are justnegligible differences in the total energyplots for the different ionic strengths ofthe media. As a consequence only the plot ofthe interaction potentials for water (Fig.5) needs to be shown.All calculated total interaction potentialdata show a minimum for the surface tosurface distance near h 2L, with L beingthe thickness of the polymer layer ofapproximately -1.4 kBT (see Table S5 and S6).Owing to the major contributing forces, vander Waals attraction, magnetic attractionand steric repulsion, being independent ofpH and ionic strength, the curves in Figure5 apply (except for the electrostaticrepulsion) for all test media in this work.The depth of the minimum (-1.4 kBT) issmaller than the Brownian motion energy of1.5 kBT 78. So the tendency of the primaryIONP to agglomerate should be weak accordingto the extended DLVO theory applied here.Obviously this is the case for all colloidalstability experiments in this work exceptfor the Elendt medium. In Elendt M7 wemeasured (Fig. 1) the fastest agglomerationprocess. This can be explained by ion-specific interactions of the polymer coatingwith Ca2+ and Mg2+ 76;77,78.It has to be kept in mind, however, thatthere are more agglomeration-sensitiveparameters like coating layer thickness,

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coating concentration on the particlesurface and the particle radius according tothe sensitivity analysis (data not shown).

In vitro testingEnzyme activityFor both isolated enzymes, acetylcholineesterase (AchE) from electric eel and yeastglutathion reductase (GR), no inhibitoryeffects of IONP could be observed under thetest conditions up to the highestconcentration tested (100 mg Fe/L). Theenzyme activity of the treated samples didnot show any significant deviation to theuntreated controls for all particleconcentrations and all replicates (data notshown).Effects on OLN-93 cells During 72 h incubation without or with IONP,the cells proliferated as indicated by thestrong increases in protein content (Fig.2a) and LDH activity (Fig. 2b). For controlcells, both parameters doubled in around 24h, which is slightly higher than thereported doubling time of these cells of 16-18 h 57. Within the incubation time of 72 hthe cells divide 3 times. Compared tocontrols (absence of IONP), chronic exposureto 100 mg Fe/L led to slightly loweredvalues for cellular protein content andcellular LDH activity per well, but thesedifferences were not significant. The smalldecrease in protein content resemblesresults that were obtained for OLN-93 cellstreated with 1 mM (55 µg/L) of iron asdimercaptosuccinate (DMSA)-coated IONP 61.Treatment of OLN-93 cells with PVP-coatedIONP even in a concentration of 100 mg/L didhardly alter the normal cell morphology anddid not compromise cell viability asindicated by the absence of any increases inthe extracellular LDH activity and in thenumber of PI-positive cells (data notshown). In addition, no increase in theamounts of cellular ROS or in the number ofROS-positive cells was observed for PVP-coated IONP-treated cells (data not shown).PVP-coated IONP are thus not acutely toxicto OLN-93 cells. This is in line withprevious reports on the resistance of thesecells towards various extracellular ironsources including IONP 61.To test for potential consequences of a

treatment with PVP-coated IONP on the cellmetabolism, OLN-93 cells were incubated for48 h without or with the particles invarious concentrations and several cellularand metabolic parameters were determined.These conditions did not significantly alterthe cell viability, but at 100 mg Fe/L the

specific lactate content released from thecells within 48 h was significantlydecreased by 20% for cells exposed to PVP-coated IONP compared to control cells (Table2). In contrast, the specific cellular GSxcontent of the cells was significantlyincreased by about 15% in cells treated with100 mg Fe/L compared to controls, while thespecific content of GSSG in the cellsremained low under all conditionsinvestigated (Table 2). The latter suggeststhat the iron accumulated from the particlesdoes not cause any oxidative stress thatwould be able to shift the GSSG to GSH ratioin the cells. This view is supported by theabsence of any increase in cellular ROSproduction. The increase in the specificcontent of GSH may indicate that PVP-coatedIONP-treated cells have a strongerantioxidative potential than control cells.During incubation of the cells with 100 mg

Fe/L, the cellular iron content increasedwithin 72 h significantly from an initialvalue of 0.07 ± 0.07 µg/well (3.5 ± 3.4 µgFe/mg protein) to 0.62 ± 0.04 µg/well (4.3 ±0.9 µg Fe/mg protein) (Fig. 2c), while lowerconcentrations of PVP-coated IONP did notincrease the cellular iron content. Time andconcentration dependent iron accumulationfrom PVP-coated IONP by OLN-93 cells is inline with data for citrate-coated 58 andDMSA-coated IONP 60. However, PVP-coatedIONP appear to be a rather poorextracellular source of cellular ironcompared to DMSA-coated IONP which led afterapplication of a total iron concentration of1 mM within 48 h to specific cellular ironcontents of about 1,000 nmol/mg (56 µgFe/mg) 60. This is more than 10-fold higherthan observed for exposure to 100 mg/L(1.8 mM) iron contained in PVP-coatedparticles. A possible reason for thisdifference could be the smaller size anddifferent zetapotential (29 ± 6 nm; -5 ± 2mV) of PVP-coated IONP compared to DMSA-coated IONP (64 ± 14 nm; -26 ± 3 mV) 51.This confirms that the type of coatingand/or the zetapotential of the IONP have astrong influence of the ability of OLN-93cells to accumulate iron, as described fornanoparticle uptake by other cells 81,82. OLN-93 cells accumulate PVP-coated IONPs mostlikely by an endocytotic uptake process assuggested before for citrate-58 and DMSA-coated IONP 60;61 rather than byextracellular liberation of iron from IONPsand subsequent accumulation of iron ions. Atleast for DMSA-coated IONPs has been shownthat presence of the iron chelatordeferoxamine did not lower the strongincrease in cellular iron accumulation ofIONP-treated OLN-93 cells 61.

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In vivo testingAcute toxicity to Daphnia magna

The PVP-coated IONP had no significanteffect on Daphnia magna in standard testperiods, although after 96 h of exposure aslight increase in toxicity occurred at thehighest IONP concentration (Fig. 3). Thismight hint at possible long-term effects inD. magna and indicate that these IONP are notinnocuous per se. Ingestion is the mainuptake route for NP in D. magna 83.Accumulated in the digestive tract,particles smaller than 130 nm can pass theperitrophic membrane (PTM) of the midgut.The PTM is kind of a mesh produced by theepithelian cells to protect them and toregulate the exchange of nutrients andenzymes 84. CuO NP (31 ± 12.8 nm) 85 and goldNP (17-23 nm) 86 were shown to pass the PTMand were found between the microvilli of themidgut epithelium, but not taken up by them.After an exposure time of 24 h intercellularspaces of epithelian cells of the midgutwere found, which might indicate a beginningdestruction of the epithel. Additionally,these authors observed an unnatural increaseof bacteria/microorganisms in the midgutlumen 85,87, which are normally held back bythe PTM 88,89. All authors consider observedtoxic effects related to oxidative stressinduced by the NP due to the increasedspecific surface area.The here performed prolongation of the

test duration for testing nanomaterials wasalso applied by 90. They found an increasedimmobilization over time and showed that theneonates were inhibited in moulting.Together with our findings, these resultsshow that acute toxicity tests alone mightnot be appropriate for a risk assessment ofnanomaterials in general. We suggeststipulating chronic tests since long-termeffects seem to play a more important rolefor the risk assessment of nanomaterialsthan their acute effects.Activity inhibition of bacteria In the assay with activated sludge, thegrowth of anaerobic bacteria was almostcompletely inhibited in the positive control(0.09 mg 3,5-dichlorophenol/mg TS; Table 3).The PVP-coated IONP showed an unexpectedinhibitory potential on the bacterialcommunity, decreasing with increasingconcentration of the particles. Moststrikingly, for the highest particleconcentration (1.5 mg Fe/mg TS) even apositive effect on bacterial activity couldbe observed. For all three particleconcentrations tested the activity at theend of the exponential phase differed

significantly from the one in the untreatedcontrols.

Despite the dissimilarity in conditions(aerobic, pure culture in liquid, endpoint:inhibition of dehydrogenase activity), theassay with Arthrobacter globiformis renderedsimilar effects of IONP: The highest effectconcentration (28% inhibition) was detectedfor 0.1 mg/L IONP, followed by a lineardecrease of this effect with increasingconcentrations (Fig. 4).

These highly unusual effects, which toour knowledge have never been describedbefore for nanomaterials and sludgebacteria, might most likely be explained bythe particle properties in the incubationmedium. If one assumes a higheragglomeration rate of nanoparticles withincreasing particle concentration (e.g. 91),the fraction of nanosized particles in theincubation medium steadily decreases withincreasing nanoparticle concentration. Ifthis fraction is smaller than the dilutionfactor, the absolute amount of dispersedparticles will increase with decreasingconcentration. Hence, for the treatmentswith the lowest particle concentrations mostIONP might be bioavailable as nanosizedparticles and could be readily taken up bythe bacterial cells 92. Especially for iron-based nanomaterials the production of ROSvia the Fenton reaction is the predominantmode of toxic action 93 and was found inbacterial strains like E. coli 41.Additionally, iron oxide particles can exerttoxic effects to bacteria and yeasts (E. coliand S. cervesiae) by adsorption onto their cellsurface 94. In that study sorption and toxiceffects were more pronounced with smallerparticle sizes and absent with largeagglomerates. For the highest concentrationsof IONP in our study, strong agglomerationand precipitation of the nanoparticles canbe assumed. This would strongly reduce thebioavailability of nanosized particles inthe solution. Moreover, the large andprecipitated agglomerates might function asa crystalline matrix for biofilm formation,fostering microbial growth. As polymercoatings have been shown to be bioavailableto bacteria95, their biodegradation may havecontributed to the activity increase aswell.

However, this does not explain the muchstronger increase of activity in activatedsludge at high IONP concentrations comparedto the A. globiformis assay. First, theparticles may have served as an energysource for iron bacteria occurring inactivated sludge 96. Second, in activatedindustrial sludge (possibly containinglarger amounts of unknown contaminants), thepronounced positive effect on the bacterial

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activity might be based on the detoxifyingproperties of IONP. Fe-based nanoparticlescan efficiently inactivate organic and othercontaminants from environmental matrices 97,which may have occurred in our study aswell. On the other hand, the low particleconcentrations probably were too small tosignificantly reduce backgroundcontamination and thus the toxic effect ofthe nanoparticulate fraction disperseddominated.

A chemical explanation for the enhancedgas production at the highest particleconcentration might be that zero-valent ironis able to reduce nitrate to nitrite and N2under biodegradation conditions comparableto our test system 98. However, such effectsare very unlikely, since in that case onewould expect a different kinetic profile ofthe gas production. Our exponentialpressure-time curves with a lag phase at thebeginning (Figure S2) provide strong supportfor a dominance of biological processes.

Model scenarios. With respect to IONP in the environment,model scenarios were used for predicting thereleased amounts and environmentalconcentrations of Gd3+ and IONP MRI contrastagents (Table 4). In the following wecompare our modeled scenario data with datafrom literature. While Kümmerer and Helmersexamined the hospital effluent in Freiburgin a small population density area based ondata of 1996 99, Bau and Dulski investigatedanthropogenic Gd anomalies in river watersin Berlin in the same year 100.

1,160 kg of Gadolinum were releasedduring ambulant examinations and 484 kg byhospital examinations each year 99. 404hospital MRI devices were used in 1996compared to 703 devices in 2008. Consideringthe assumptions (see ESI), 2,536 kg of Gd3+

were released into the environment (Table4), composed of 1,690 kg release in ambulantexaminations and 845 kg Gd3+ emission fromhospitals in 2009. The increasing use ofGd3+ in the business-as-usual scenario couldlead to a more critical environmentalrelease in 2020, which is doubled to 4,814kg compared to 2009 (Table 4). The PECSurface Water shows a comparabledevelopment from 0.028 µg/L to 0.053 µg/L.In the light of a two orders of magnitudelower Gd3+ background concentration of 0.4ng/L 101 and 0.6 ng/L 100 applications of Gdcan pose additional stress to theenvironment 102;103). Despite this, only veryfew studies were conducted for acutetoxicity and none (as far as known) forchronic effects 104. The only known study fora predicted no-effect concentration (PNEC)

reports growth inhibition effects on greenalgae at 20 mg/L by the contrast agentomniscan® (gadodiamide) 105, which is veryhigh. The Gd complexes in contrast agentsprevent their biovailability, yettransformation processes in the environmentmay render highly toxic free Gd3+ 105. Theseestimated concentrations are similar to theresults from 99 who estimated the PECSurface Waterfor Gd3+ between 0.011 and 0.026 µg/L.Comparing this with 100 who estimated a max.PECSurface Water of 0.1 µg/L in densely populatedareas, it becomes obvious how calculatedvalues can vary in a regional context.

The best-case scenario assumes that ironoxide based contrast agents are an adequatesubstitution of Gd3+ based contrast agentsand would hold a market share of 25% in2015. Almost 260 kg of iron oxide contrastagents could replace 900 kg of Gd3+ in MRIuse and balance the increasing PECSurface Waterof Gd3+ to 0.026 µg/L, respectively. If themarket share rises until 2020, iron oxidebased agents could reduce the Gd3+ releaseby about 300 kg compared to 2015.Consequently a slight PECSurface Water decreaseto 0.023 µg/L could be postulated (Table 4).

Applying the Swiss precautionary matrix 75

to the IONP as possible contrast agent forMRI resulted in rather little precautionaryneed (WG, WWC, C, E) (Fig. 6a). In theliquid media scenarios (PL, UL), values forthe precautionary need were just above thelimit below which basically no “nanospecificaction is needed” (20 points, 75). The drypowder scenarios (PD, UD) renderedrelatively higher values which, however, didnot reach more than one tenth of the highestpossible values.

Also for the application of IONP forremediation purposes (Fig. 6b) the matrixrendered at most 10% of the possible maximumvalues (ML, MD). nZVI is generally morereactive than IONP and thus revealed ahigher precautionary need (ZVL, ZVD), evenreaching the maximum in the dry powderscenario (ZVD) (Fig. 6b).

The main results regarding potentialhuman health effects and exposure correspondquite well with the published literature(see introduction). The precautionary needfor the environment was generally higherwhen applying the matrix to the remediationcases, particularly when using nZVI as drypowders. This is, however, not realisticsince nZVI is usually applied as dispersionwith as little oxygen contact as possible toavoid oxidation. Some studies 41-43 suggestthat IONP may indeed be ecotoxic at veryhigh particle concentrations (which may bethe case in remediation scenarios). Heresome weakness of the precautionary matrixbecomes evident: the amount of particles

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entering the environment has relativelylittle effect on the final outcome, even ifit is as huge as in realistic remediationscenarios. The by far most decisiveparameter with respect to the totalprecautionary need is the availability ofthe nanoparticles (free, suspended or insolid matrices), which may be sensible withregard to direct human health risks.Concerning environmental risks, however, theamounts (potentially) entering theenvironment are probably at least asimportant as the availability and should,therefore, be weighted higher in theprecautionary matrix.

ConclusionsThe PVP-coated iron oxide nanoparticlessynthesized in this study were very stableat a concentration of 100 mg/L in allinvestigated media. Even if the particlediameter increased up to 232 nm, as in thecase of Elendt M7, no precipitation wasfound. Since PVP is a standard coating formany medical applications with a possiblerelease into the environment, our stablecolloidal solutions of PVP coated iron oxidenanoparticles are perfect for investigatingthe influence of iron oxide nanoparticles onbiological and environmental systems.The calculated particle colloidal

stability was in good agreement withexperimental results except for Elendtmedium because of ion specific interactionsof calcium and magnesium cations with theparticles in this work. That means theapplication of the extended DLVO theoryincluding magnetic attraction and stericrepulsion here shown is the right choice tomodel these IONP interaction potentials inmedia containing ions without specificinteractions with particles. The coatingthickness and concentration on the particlesurface as well as the particle radius atstart seem to be important factors instabilizing IONP dispersions. However,specific ion interactions should be includedin the model to correctly estimate colloidalstability in the presence of these ions inthe system.In a concentration range up to 100 mg Fe/L

the particles appear to be highly compatiblewith the proliferation and the metabolism ofOLN-93 cells. Also the enzymatic assays withGR and AchE rendered no functionalinhibition with respect to oxidative stressor neuronal function. Even when the exposure time was doubled

compared to the standard test, acutetoxicity tests only revealed a slight,insignificant increase in immobilization of

Daphnia magna at the highest testconcentration (100 mg Fe/L). However, acutetoxicity tests might not be appropriate fora risk assessment of nanomaterials ingeneral, since long-term effects likedissolution kinetics, formation andbreakdown of agglomerates or the particles’interaction with environmental matricesleading to surfaces changes seem to play asignificant role in the toxicologicalbehaviour of nanoparticles.Both microbial inhibition assays showed a

high ecotoxicological potential of the IONP,especially at low concentrations. Furtherstudies on the agglomeration behaviour ofthe particles under these environmentallyrelevant conditions are needed to elucidatewhether the toxic effects on bacteria aredue to nano-specific effects as our studysuggests.Additionally, it should be kept in mind

that for a complete hazard assessment long-term data and toxico-kinetic models shouldbe included, since the processing of theparticles in organisms and dissolution ofthe particles may lead to an elevated hazardpotential, as described e.g. by 106.The rough PEC estimation has shown that

iron oxide contrast agents have thepotential to reduce environmental damage byreplacing the Gd based contrast agents. Withrespect to risk management, our scenariosclearly indicate that IONP should preferablybe produced, handled, used, and disposed insuspended form, since potential exposure andhealth risks are significantly higher fordry powder (inhalation). Lowering theparticle stability, e. g. from “month” to“weeks”, would result in significantly lowerprecautionary needs, i.e., lower potentialrisk. Another consequence is the necessityfor nanomaterial specific waste treatment ordisposal. This would lower the potentialinput into the environment and thusprecautionary need. As demonstrated for theremediation scenarios, the Swissprecautionary matrix in its present formmight need some revision with respect to theimpact of expected amounts in theenvironment on the final outcome.In summary, the broad approach of our

study has shown that at least PVP-coatedIONP are not intrinsically green. Existingtest and modeling procedures mightunderestimate potential risks of IONP in theenvironment, especially hazards tomicroorganisms (which do not belong to thestandard testing strategy). Forprecautionary reasons, particularly theirapplication for site remediation purposesshould be well considered until more data isavailable.

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AcknowledgementsThis work was funded by Hans-Böckler-Stiftung(HBS), Düsseldorf/Germany, by Verband derChemischen Industrie (VCI), Frankfurt, M./Germanyand by the University of Bremen/Germany.Furthermore, it was funded by the State ofBremen/Germany, Ökologiefonds, FörderprogrammAngewandte Umweltforschung supported by theEuropean Fund for Regional Development 2007-2013. The authors thank Prof. C. Richter-Landsberg(University of Oldenburg, Germany) forkindly providing OLN-93 cells. They alsothank Karin Nitsch for linguisticcorrections and Antje Mathews for her greatsupport of revising the manuscript.

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Notes and references‡ The test design was adapted to the specificcharacteristics of nanoparticles. Theseadaptations give the ability to work more cost-effective. Saving animals and substance allows toperform more independent tests.

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