and Composition-Dependent Toxicity of Synthetic and Soil-Derived Fe

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Size- and composition-dependent toxicity of synthetic and soilderived Fe oxide colloids for the nematode Caenorhabditis elegans Sebastian Höss, Andreas Fritzsche, Carolin Meyer, Julian Bosch, Rainer Meckenstock, and Kai Totsche Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es503559n • Publication Date (Web): 01 Dec 2014 Downloaded from http://pubs.acs.org on December 3, 2014

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Environmental Science & Technology

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Size- and composition-dependent toxicity of synthetic and soil-derived Fe oxide colloids

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for the nematode Caenorhabditis elegans

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Sebastian Höss1,2, *, Andreas Fritzsche3, Carolin Meyer4, Julian Bosch4,

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Rainer U. Meckenstock4,5 and Kai Uwe Totsche3

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1

Institute for Biodiversity – Network (IBN), Nussbergerstr. 6a, 93059 Regensburg,

8

Germany

9

2

10

3

Ecossa, Giselastr. 6, 82319 Starnberg, Germany

Insitute of Geosciences, Friedrich-Schiller-University Jena, Burgweg 11, 07749 Jena,

11 12

Germany 4

Institute of Groundwater Ecology, Helmholtz Center for Environmental Health, Ingolstädter

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Landstr. 1, 85764 Neuherberg, Germany 5

Aquatic Microbiology, University of Duisburg-Essen, Universitätsstr. 5

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45141 Essen, Germany**

16 17 18 19

* corresponding author:

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Sebastian Höss

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Email: [email protected]

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Phone: +49-8151-5509172

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FAX: +49-8151-5509173

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** present address

1 ACS Paragon Plus Environment


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Abstract

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Colloidal iron oxides (FeOx) are increasingly released to the environment due to their use in

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environmental remediation and biomedical applications, potentially harming living organ-

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isms. Size and composition could affect the bioavailability and toxicity of such colloids.

29

Therefore, we investigated the toxicity of selected FeOx with variable aggregate size and var-

30

iably-composed FeOx-associated organic matter (OM) towards the nematode Caenorhabditis

31

elegans. Ferrihydrite colloids containing citrate were taken up by C. elegans with the food

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and accumulated inside their body. The toxicity of ferrihydrite, goethite and akaganeite was

33

dependent on aggregate size and specific surface area, with EC50 values for reproduction

34

ranging from 4-29 mg Fe l-1. Experiments with mutant strains lacking mitochondrial superox-

35

ide dismutase (sod-2) showed oxidative stress for two FeOx and Fe3+-ions, however, revealed

36

that it was not the predominant mechanism of toxicity. The OM composition determined the

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toxicity of mixed OM-FeOx phases on C. elegans. FeOx associated with humic acids or cit-

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rate were less toxic than OM-free FeOx. In contrast, soil-derived ferrihydrite, containing pro-

39

teins and polysaccharides from mobile OM, was even more toxic than OM-free Fh of similar

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aggregate size. Consequently, the careful choice of the type of FeOx and the type of associat-

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ed OM may help reducing the ecological risks if actively applied to the subsurface.

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Introduction

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Chemical or microbial degradation of organic groundwater contaminants can be stimulated by

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the injection of dispersed iron-based nanomaterials (Fe-NM) into contaminated aquifers 1. In

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consequence, Fe-NM hold a large potential for cost- and resource efficient in situ remediation

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of contaminated groundwater aquifers 2. Dispersed iron oxides (FeOx), i.e. FeOx colloids, are

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highly reactive Fe-NM that exceed the already distinct reactivity of non-colloidal FeOx3,4. In

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addition to nitrate, Mn(IV) oxides and sulfate, FeOx colloids may also serve as highly availa-

50

ble electron acceptors

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contaminants. Furthermore, FeOx colloids are synthesized and applied in cleaning-up sedi-

52

ments and drinking water by binding toxic metals

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medical purposes 9. Consequently, synthetic FeOx colloids will be increasingly released into

54

the environment, posing a potential risk for the biota of groundwater and surface water eco-

55

systems. Nanoparticles (NP) are assumed to be more hazardous to organisms than larger-sized

56

particles of the same material, because their higher surface-to-mass ratio potentially causes a

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higher biological activity, e.g. inflammatory and pro-oxidant activity

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sizes for ecological effects of NP still have to be defined, as common fixed values (e.g. 100

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nm) as benchmark for “nano” and “not-nano” might not be appropriate

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properties governing the toxicity of FeOx colloids are still obscure, because studies on the in

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vivo toxicity of FeOx colloids on multicellular organisms are scarce 12.

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Nematodes are one of the most abundant and species-rich organism group and occupy key

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positions in the food web of soils and aquifers

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group for risk assessments in these environments

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elegans is not a typical representative of groundwater nematodes, it can be found in freshwa-

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ter habitats (e.g.

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species (e.g. species of Rhabditis

17

5

for microbial anaerobic biodegradation of hydrocarbon groundwater

6,7

13–15

, in wastewater treatment 8, and for bio-

10

. However, threshold

11

. Yet, the particle

. Therefore, they are a relevant organism 16

. Although the species Caenorhabditis

) and is taxonomically closely related to groundwater dwelling nematode 18

). C. elegans is increasingly recognized as a suitable test 3

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organism to assess the toxicity of chemicals and environmental samples

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toxicity tests with C. elegans (ISO 10872 21) have been successfully applied for assessing the

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toxicity of aqueous solutions 22,23, soils 24,25 and sediments 26,27.

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In the environment, inorganic colloids interact with organic matter (OM), modifying their

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surface properties and thus also their geochemical behavior and ecotoxicological properties 28.

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For medical purposes, particles are coated with OM to enhance their uptake efficiency into

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cells 29. In contrast, humic substances, which are part of OM in soils and sediments, reduced

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the toxicity of nanoparticles

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OM composition 32,33. However, the cited studies do not refer to FeOx colloids pointing to the

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gap in knowledge for these minerals. Despite their common use in laboratory studies, humic

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and fulvic acids also are of limited use as equivalents for OM, which is actually available for

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interactions with (colloidal) minerals in soils and sediments. This is reasonable considering

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the differences in composition of OM from the aqueous phase of natural porous media, i.e.

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mobile OM, compared to OM, which is obtained by alkaline extraction or retention on hydro-

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phobic resins, i.e. humic and fulvic acids 34.

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In this study, we investigated the toxicity of FeOx, i.e. ferrihydrite, goethite and akaganeite,

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towards the nematode C. elegans. Ferrihydrite and goethite are the most abundant FeOx in

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soils and sediments of temperate climate zones 4. While akaganeite is less abundant in nature,

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it represents a synthesized FeOx, which is introduced into soils and sediments for remediation

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purposes 35. These FeOx are known to persist in colloidal state in natural porous media

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We investigated the dependence of FeOx toxicity on i) variable FeOx aggregate sizes and ii)

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variable types of FeOx-associated OM (citrate, humic acids, mobile OM from soil). We fur-

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thermore focused on the fate of ferrihydrite colloids after being ingested by C.elegans and the

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role of FeOx-mediated oxidative stress. We aimed for answering the following questions: (1)

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Does C. elegans incorporate and accumulate FeOx colloids and can toxic effects then be ex-

30,31

19,20

. Standardized

, while mitigation of toxicity was dependent on the actual

35–37

.


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plained then by oxidative stress? (2) Is FeOx toxicity on C. elegans influenced by aggregate

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sizes and associated organic compounds? (3) Are synthetic FeOx colloids more toxic than soil

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derived FeOx colloids?

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Materials and Methods

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Production of synthetic FeOx. Low crystalline, i.e. 2-line ferrihydrite (Fh) was prepared

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by titration of 0.1 M Fe(NO3)3•9H2O (A.C.S. grade, Sigma-Aldrich) to pH = 7.0 with 0.1 M

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KOH (Merck)

38

. Residual ions were removed by washing the precipitate 6 times with ul-

100

trapure water (R=18.2 MΩ, 4 ppb TOC, Millipore Elix + Milli-Q Advantage 10A), which was

101

used as solvent also for the preparation of all other synthetic FeOx. The last 3 washes includ-

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ed centrifugation (4000 g, 30 min., 4 °C, AvantiJ-E centrifuge, JA-10 rotor, Beckman Coul-

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ter). The obtained pellet was re-suspended and stored. Owing to proceeding Fh aggregation

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during storage, two different mean aggregate sizes were obtained from this preparation

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(Fh_small, Fh_med; Table 1). Micron-sized aggregates of 2-line Fh (Fh_large; Table 1) were

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synthesized with 0.4 M FeCl3 • 6H2O (reagent grade, Sigma-Aldrich), which was titrated to

107

pH 7.0 with 1 M NaOH (ACS grade, Sigma-Aldrich)

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least 5 times until the supernatant remained clear. Fh with citrate (Fh_citrate) was produced

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by dissolving ferric citrate (ACS grade, Sigma) and adjusting pH to 8.0 with 10 M NaOH

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under strong stirring 40. The resulting Fh colloids were concentrated by several cycles of cen-

111

trifugation and re-suspension until no further increase in electric conductivity could be ob-

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served in the suspension.

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A 1 M nanogoethite suspension (Goe, Helmholtz Center for Environmental Health, Germany)

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was prepared (patent pending) and dialyzed (Zellutrans T2 dialysis tubes, molecular weight

115

cutoff (MWCO) 8-10 kDa, Roth) against ultrapure water to remove residual ions. For the pro-

116

duction of akaganeite (Aka), 0.37 M FeCl3 • 6H2O (ACS grade) was heated (60 °C) for 10 h

117

41

39

. The precipitate was washed for at

. The cooled suspension was dialyzed in cellulose bags (MWCO: 3.5-5.0 kDa, Roth) against 5 ACS Paragon Plus Environment


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water (pH = 4, adjusted with 0.5 M HCl). All iron oxides were stored at 4 °C in the dark. Au-

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toclaving was omitted to preserve the crystal structure.

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Before adsorption of humic acid (HA), FeOx suspensions were sonicated under constant stir-

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ring for 2 hours (UP50H Sonifier, Meinhardt UltraschallTEC). A dilute solution (1g l-1) of

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HA sodium salt (Sigma Aldrich) was titrated with 1 M HCl to pH = 7.0 and stirred overnight

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at room temperature. After centrifugation for 20 minutes (20 °C; 4000 g), the supernatant was

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used for FeOx coating.

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Production of soil-derived FeOx. The detailed procedure to produce organo-mineral FeOx

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colloids is given SI 1. Briefly, FeOx precipitated in soil column effluents due to oxidation of

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Fe2+, which was mobilized from the soil owing to the reduction of pedogenic Fe(III) by au-

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tochthonous microbial communities. We expect such organo-mineral FeOx to form at anoxic-

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oxic interfaces in soils. A subset of the FeOx-containing soil effluent was dialyzed (1000

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kDa; Spectra/Por Float-A-Lyzer G2, Spectrum Laboratories) against ultrapure water. Dia-

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lyzed and non-dialyzed soil effluents were used in the toxicity assays to allow for differentia-

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tion between FeOx-mediated effects and effects potentially arising from the coexistent efflu-

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ent compounds (ions, low molecular OM).

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FeOx characterization. Dialyzed FeOx suspensions were characterized with X-ray diffrac-

135

tion (XRD), Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy

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(SEM), dynamic light scattering (DLS) and laser Doppler velocimetry (LDV). XRD and FTIR

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required freeze-drying (Alpha 1-4 LSC, Christ). XRD (D8 Advance; Bruker) was conducted

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on Si (911)-holders with Cu-Kα radiation (40 kV, 40 mA, 10-80 °2θ, 0.02 °2θ steps á 1 s).

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FTIR spectra (Nicolet iS10 spectrometer; Thermo Fisher Scientific) were recorded in trans-

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mission mode (16 scans per spectrum, resolution: 4 cm-1). The FeOx were ground, mixed with

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KBr (FTIR spectroscopy grade, Merck) and pressed to pellets. For SEM, the dialyzed suspen-

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sion was air-dried on a Si wafer. Analysis was conducted with an ULTRA PLUS field emis6 ACS Paragon Plus Environment

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sion scanning electron microscope (Zeiss). Aggregate areas were calculated with the software

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ImageJ (v. 1.47) from secondary electron (SE) images on 3 wafer sections (Fh_large: 1 wafer

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section). In consideration of the lower resolution limit of the SEM device, particle areas 1.96 (two-tailed test)

246

54

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Results and Discussion

[Equ. 1]

s 2 log ECx N 2 + s 2 log ECxsod

.

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Fe accumulation in C. elegans. Immediately after exposure, relatively high Fe concentra-

249

tions were measured in the nematodes exposed to Fh_citrate (Figure 1: 0h post exposure).

250

However, these organisms still contained considerable amounts of Fe in the intestinal lumen,

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as well as attached to their cuticle, masking the truly internalized Fe concentrations in the

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nematodes’ tissue. After 2 h of defecation, the nematodes cleared their gut from the Fh_citrate

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colloids by feeding on fresh bacteria, resulting in a 50% loss of Fe taken up with the food

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(Figure 1). Disposal of the surface-attached Fe with the cuticula (after 8 h) caused an addi-

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tional 80% reduction of nematode-associated Fe. The high variability after 2 h is very likely

256

caused by variable molting rates between the replicates. The presence of nematode-associated

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Fe after complete molting, i.e. after 8 h, and the good agreement between the replicates (Fig-

258

ure 1) indicate a significant and quantitatively reproducible internalization of Fe in C. ele-

259

gans’ tissue, where toxicity is assumed to occur. Neither Fe attached to the cuticle, nor Fe

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located in the intestinal lumen was considered to substantially contribute to Fe-mediated tox-

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icity in the nematodes: (1) The main uptake route for metals into the tissue of C. elegans oc-

262

curs via the gut, rather than via the cuticle

22,55

263

served for TiO2 nanoparticles in C. elegans

56

. (2) Blockage of the gut by particles as ob-

, which might have deleterious effects on the



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nematodes, was not observed under the light microscope (400-fold magnification; data not

265

shown) after exposure to FeOx.

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Fh-citrate exposed organisms showed a significantly elevated Fe concentration in the tissue

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(mean ± ; 0.268 ± 0.047 µg Fe mg-1 wet weight (ww)) compared to control organisms (0.059

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± 0.016 µg Fe mg-1 ww) that were not exposed to Fh_citrate (p < 0.01, t-test, n = 3; Figure 1).

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Related to the exposure medium (28 mg Fe l-1), Fe was accumulated by C. elegans by a factor

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of 7.5 (± 1.7). In vitro studies showed that FeOx nanoparticles (NP) can enter endothelial cells

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even without protein-coated vesicles, suggesting a micropinocytotic process

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translocated in the uterine area or along the digestive organ of C. elegans

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could not be proved that the accumulated Fe still represented intact Fh_citrate colloids. In the

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gut of the nematodes, where pH can range between 6.0 (anterior pharynx) and 3.6 (posterior

275

intestine)59, FeOx might partly dissolved due to protonation. From a kinetic point of view,

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however, the very short exposure time of FeOx in acidic parts of the intestine (< 2 min

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makes a quantitative dissolution of FeOx unlikely. Moreover, uptake experiments with ionic

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Fe3+ showed only slightly, not significantly elevated Fe concentrations after 6 h exposure

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(0.108 ± 0.031 µg Fe mg-1 tissue ww) compared to the control (0.052 ± 0.018 µg Fe mg-1 tis-

280

sue ww; p > 0.05; t-Test, n = 3). In contrast, Fe tissue concentrations after Fh_citrate colloid

281

exposure at the same exposure concentration was significantly elevated compared to the con-

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trol (0.185 ± 0.026 µg Fe mg-1 ww; p < 0.05; t-Test, n = 3). This indicated that only a minor

283

part of Fe was accumulated from co-occurring ionic Fe3+. However, it has to be noted that C.

284

elegans is able to effectively concentrate food particles by pharyngeal pumping 48. Therefore,

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internal exposure might have been higher after colloid FeOx compared to ionic Fe3+ exposure.

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A partial dissolution of concentrated Fh_citrate in the acidic environment of the gut might

287

have allowed for a minor transfer of ionic Fe3+ from the gut to the tissue.

57

. Ag-NP were

33,58

. However, it

60

),


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FeOx toxicity The average minimal detectable difference (MDD; mean ± ) in all tests

289

(n=28) was 18.5 ± 7.7% for effects on the reproduction of C. elegans. Therefore, first substan-

290

tial effects were expected at ~20% inhibition of reproduction. Besides the median effect con-

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centration (EC50), EC20 was therefore selected as an appropriate endpoint for describing the

292

toxicity at a low effect level. The toxicity of the various OM-free and organo-mineral FeOx

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on the reproduction of C. elegans ranged from EC20 = 2.2 - >212 mg Fe l-1 and from EC50 =

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4.0 - >212 mg Fe l-1 (Table 2). In comparison, ionic Fe3+ showed EC20 and EC50 values of

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8.3 and 12.1 mg Fe l-1, respectively (Table 2). This points to a comparably high susceptibility

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of C. elegans to ionic Fe3+, given the relatively low internalized concentrations in the nema-

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todes’ tissue. Related to the exposure concentrations in the test medium, toxicity of ionic Fe3+

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to C. elegans was comparable to Fh_small and Fh_medium, however lower than Aka and

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Fh_soil. Co-occurrence of FeOx and ionic Fe3+ could therefore result in biased EC20 and

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EC50 for FeOx. However, we assume only negligible concentrations of ionic Fe3+ in the FeOx

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suspensions in K-medium, which is in equilibrium with goethite and ferrihydrite, as low as

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26.5 ng Fe l-1 and 13.7 µg Fe l-1, respectively (pH = 5.5, pE = 15.1; PHREEQC 3.1.4 with

303

minteq.v4-database). As discussed above, the short residence time of FeOx colloids in the

304

acidic environment of the nematodes’ gut makes a quantitative dissolution into Fe3+ ions un-

305

likely. A partial contribution of Fe3+ ions to the observed FeOx colloid toxicity, however,

306

cannot be excluded. Nevertheless, even a complete dissolution to Fe3+ could not explain the

307

comparatively higher toxicity of Aka and Fh_soil (Tab. 2; Fig. 2).

308

There exist only few studies about the toxicity of FeOx colloids on aquatic or soil organisms

309

to which the results of the present study can be compared. Wu et al. 12 evaluated the sensitivi-

310

ty of C. elegans to maghemite-NP coated with dimercaptosuccinic acid (DMSA) using vari-

311

ous sublethal toxicity endpoints. With comparable test conditions (exposure from J1 juveniles

312

to adults) they found first significant effects on brood size at 0.5 mg Fe l -1, which indicated an 13 ACS Paragon Plus Environment


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even higher toxicity compared to the present study. However, it is not clear, if and to what

314

extent the DMSA coating contributed to the toxicity. The protozoan Paramecium multimicro-

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nucleatum was acutely affected by relatively low concentration of commercially available

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nano-sized FeOx (Sigma-Aldrich; LC50: 0.81 mg l-1)

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however, did not respond to FeOx (maghemite) nanoparticles up to a concentration of 700 mg

318

l-1 62.

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With respect to the OM-free Fh, the toxicity clearly decreased with increasing aggregate size,

320

with significant lower EC50 values in Fh_small and Fh_med compared to Fh_large (Table 2).

321

According to SE images, the size of the OM-free Fh aggregates agreed with the nominal clas-

322

sification, i.e. small, medium and large (Table 1). However, the OM-free Fh-aggregates fur-

323

ther aggregated at sufficient presence of electrolytes, i.e. in the K-medium. This was indicated

324

by interferences of larger aggregates on DLS measurements (Table 2). Since Fh aggregates

325

also remained dispersed in the K-medium, we assume this aggregation to not quantitatively

326

affect the Fh aggregates. Despite the increased mean aggregate sizes of all OM-free synthetic

327

FeOx, we therefore expect that independent from actual values the nominal classification of

328

the Fh aggregates (small, medium, large) remains also valid in the K-medium. The synthetic

329

OM-free Fh contained traces of goethite (Goe) and hematite (SI 2). Since all types of OM-free

330

Fh were affected likewise, we attribute this to the synthesis and not to the different periods of

331

storage. Besides the identical mineral composition, all OM-free Fh had a net-positive surface

332

charge, which is indicated by positive UE (Table 1). Consequently, shifts in EC50 values can

333

be referred to shifts in Fh aggregate sizes since this is the variable property among these

334

FeOx. It has to be noted that actual exposure concentrations for C. elegans might have been

335

higher for larger than for smaller FeOx, because larger FeOx aggregates settle to a larger ex-

336

tent to the bottom of the test vial, where the nematode fed on the bacteria. Thus, the toxicity

337

of the larger FeOx aggregates was overestimated compared to the smaller, dispersed FeOx.

61

. The bacterium Escherichia coli,


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This intensifies the aggregate-size effect for the toxicity. Living organisms are assumed to be

339

harmed to a greater extent by NP compared to larger-sized particles of the same material, be-

340

cause their higher surface to mass ratio potentially might cause also a higher biological activi-

341

ty. NP of Al2O3, ZnO and TiO2 were more toxic in terms of reproduction of C. elegans than

342

the corresponding non-NP

343

cle/aggregate sizes, the smallest FeOx aggregates in this study, i.e. the akaganeite colloids

344

(Aka) (Table 1), were also the most toxic FeOx (Table 2). However, considering the lower

345

toxicity of the Goe colloids (Table 2), it is obvious that aggregate size was not the exclusive

346

parameter controlling the FeOx toxicity on C. elegans. Although the aggregate size of Goe

347

was similar to Fh_small (Table 1), EC50 were significantly higher than Fh_small and Fh_med

348

(Table 2). Here, the specific surface area (SSA: total surface area per mass unit) has to be

349

considered as additional factor influencing the FeOx toxicity. In our study, Goe colloids had a

350

considerably lower SSA compared to Fh_small despite similar aggregate sizes (Table 1).

351

Higher SSA can be aligned with a higher number of reactive surface sites per unit mass. The

352

mineral surface (particularly of minerals containing transition metals such as iron) can cata-

353

lyze the reduction of O2 via redox reactions and thus induce oxidative stress in living cells by

354

the formation of reactive oxygen species (ROS)

355

duction of ROS in C. elegans after exposure to DMSA-coated maghemite-NP, while sod-2

356

and sod-3 mutants produced ROS and exhibited behavioral response at significant lower con-

357

centrations (0.01 mg Fe l-1) than the wild-type (0.1 mg Fe l-1), clearly indicating oxidative

358

stress. Besides Fe3+ ions, only Fh_citrate and Fh_large exhibited a significantly increased in-

359

hibition of the reproduction of the oxidative-stress hypersensitive mutant strain sod-2 com-

360

pared to wild type C. elegans (Table 2). However, with EC50sod/EC50N2 ratios of 0.73 (Fe3+),

361

0.64 (Fh_citrate) and 0.77 (Fh_large), the Fe ions and FeOx showed a less distinct oxidative

362

stress than the positive control paraquat (ECxsod/ECxN2 ratio: < 0.1; Table 2). Apparently,

63

. In agreement with increasing toxicity with decreasing parti-

64

. Wu et al.

12

found a dose-dependent pro-



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oxidative stress only partly contributed to FeOx toxicity towards C. elegans and could not

364

explain the link between FeOx toxicity and aggregate size and SSA. Moreover, the oxidative

365

stress observed for Fh_citrate and Fh_large might have been mediated by Fe3+ co-occurring in

366

the acidic part of the nematodes’ intestine.

367

Colloid-associated OM substantially influenced the toxicity of FeOx, while the OM composi-

368

tion was crucial for the toxic level of the colloids. According to XRD, humic acid-associated

369

goethite (Goe_HA) was of identical structure compared to OM-free Goe (SI 2). Fh_HA com-

370

prised low crystalline, i.e. 2-line Fh only, while Fh_citrate contained traces of Goe (SI 2).

371

Contrary to Goe, the OM-free and organo-mineral Fh suspensions consequently do not com-

372

pletely agree with each other with respect to mineral structure. However, given the predomi-

373

nance of intrinsically weak 2-line Fh-related X-ray reflexes in the diffractograms, Fh was still

374

the quantitatively dominant FeOx. Besides identical or at least very similar mineral structures

375

in the treatments with OM-free and OM-containing Goe and Fh, respectively, all treatments

376

exhibited nearly identical aggregate size distributions (Table 1). We therefore assume that

377

differences in toxicity can be attributed to the type of FeOx-associated OM. Fh_citrate,

378

Fh_HA and Goe_HA were significantly less toxic compared to the corresponding OM-free

379

colloids (Table 2). However, this effect was much more pronounced for HA compared to cit-

380

rate. In contrast to HA and citrate, mobile OM from soil enhanced the toxicity of Fh. Com-

381

pared to synthetic Fh, Fh_soil inhibited 87-99% of the reproduction of C.elegans at concen-

382

tration as low as 15-30 mg Fe l-1 (Figure 2). For all organo-mineral FeOx colloids, the pres-

383

ence of OM resulted in a net-negative surface charge, which is reflected by negative UE (Ta-

384

ble 1). Surface charge of nanoparticles can influence their toxicological action, with positively

385

charged particles being incorporated in cells to a larger extent and showing a higher cytotoxi-

386

city compared to negatively charged particles (e.g. Au particles 65). For C. elegans CeO2 par-

387

ticles with a positively charged coating also were found to be more toxic than those with a 16 ACS Paragon Plus Environment

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negatively charged coating. However, considering the different effects of citrate, HA and soil-

389

derived OM on FeOx toxicity, this consistent OM-mediated inversion of FeOx surface charge

390

cannot solely explain the altered interactions between organo-mineral vs. OM-free FeOx col-

391

loids with C.elegans. The considerably lower toxicity of Fh_HA compared to Fh_citrate could

392

be explained by the higher content of aromatic compounds in HA, that more efficiently de-

393

creased toxicity of FeOx than citrate. This is in agreement with assumptions of Lee et al.

394

that hydrophobic coatings reduce the bioavailability and thus also the toxicity of nanoparti-

395

cles. However, Yang et al. 33 found no difference between the uptake of Ag-NP in C. elegans

396

in presence or absence of fulvic acids. Another possible explanation is that HA occupied the

397

bioactive sites of the NP that had been responsible for the toxic effects 31. Also CeO2 toxicity

398

on C. elegans was significantly reduced by the presence of HA 30.

399

Although soil-derived OM also contains aromatic compounds, the composition is fundamen-

400

tally different compared to HA. This arises by the type of extraction, i.e. extraction from soil

401

with low ionic solutions at neutral pH (SI 1) vs. strong alkaline extraction from solids or ex-

402

traction with resins from liquids, respectively 34. Consequently, the soil effluent OM contains

403

compounds, which are sufficiently polar at neutral pH to permit sufficient hydratization and

404

thus solubilization into the aqueous phase. Such compounds are for instance proteins and pol-

405

ysaccharides, which are abundant in soil effluent OM (SI 3) and accordingly in Fh_soil (SI 6)

406

but not in HA (SI 3). We exclude interferences on toxicity of Fh_soil by (in)organic effluent

407

constituents that were not associated with Fh. Effluent dialysis, which removed ions and small

408

molecules but not Fh_soil from the effluent, only slightly decreased the toxicity (SI 7). This

409

was caused by a reduction of effluent Fe concentrations after dialysis (Table 1) due to settling

410

Fh aggregates, which could not be completely recovered from the dialysis tube. Toxic effects

411

of soil effluent OM alone could be excluded, because tests with dialyzed soil effluent, which

412

contained the same mobile OM but was free of Fh (SI 1), revealed no toxicity, but even

32



Page 18 of 33

413

stimulated the reproduction of C. elegans (SI 7). Therefore, we assume that the pronounced

414

presence of hydrophilic compounds in Fh_soil-associated OM, i.e. proteins and polysaccha-

415

rides, increased the bioavailability of Fh_soil at target sites of C.elegans (e.g. membranes),

416

which could result in higher toxicity compared to Fh_HA and Fh_citrate.

417

Environmental implications. If Fe oxide (FeOx) colloids are introduced into the environ-

418

ment for remediation purposes

419

need to be assessed to avoid sustained damage to ecological functions. Our study showed that

420

for C. elegans, soil-derived ferrihydrite colloids were as toxic as the most toxic synthetic

421

FeOx colloids (akaganeite). Unless concentrations of synthetic FeOx colloids do not highly

422

exceed those of in situ formed colloids, synthetic FeOx do not a priori pose an additional

423

hazard to nematodes. Concentrations of FeOx >10 mg Fe L-1 are not likely to be mobile over

424

long distances in soils (decimeter scale). However, natural FeOx colloids are locally abundant

425

in such concentrations at anoxic-oxic interfaces, where ionic Fe2+ oxidizes and precipitates as

426

FeOx. Thus, if high amounts of synthetic FeOx colloids need to be applied to the subsurface,

427

e.g. for remediation purposes, it is advised to use FeOx with a minimal toxic potential, e.g.

428

goethite colloids. Coating with humic acids (HA) would further decrease the toxic potential of

429

FeOx, at least for nematodes. Additionally, we expect that occupation of reactive FeOx sites

430

with HA will decrease the probability that mobile organic matter (OM) from soils and sedi-

431

ments increases the FeOx toxicity due to association with FeOx surfaces and thereby enhanc-

432

ing the FeOx bioavailability for nematodes. On a long term basis, the accumulation of signifi-

433

cant amounts of FeOx by C.elegans might result in the transfer to organisms of higher trophic

434

levels feeding on nematodes in terrestrial or aquatic food webs.

435

The opposite effects of HA and soil effluent OM, when associated with Fh, on C. elegans also

436

emphasizes the crucially different functions of such differently composed OM. Adverse influ-

437

ence of OM on Fh toxicity amplifies these differences, where comparably high aromaticity of

2,6

the risks of harmful effects on the subsurface organisms


Page 19 of 33


438

HA decreases the Fh toxicity and hydrophilic compounds in soil effluent OM increases Fh

439

toxicity.

440

Associated Content

441

Supporting Information

442

Additional data on FeOx characterization and toxicity is compiled as cross-referenced

443

throughout the manuscript. This material is available free of charge via the Internet at

444

http://pubs.acs.org.

445

Author Information

446

Corresponding Author: *Sebastian Höss, email: [email protected]

447

Acknowledgments

448

This study was supported by the German Ministry of Education and Research (BMBF joint

449

project NanoSan; Grant No. 03X0085). The gifts of Caenorhabditis elegans (strains N2 and

450

RB1072) and Escherichia coli (strain OP50) from the Caenorhabditis Genetic Center, which

451

is supported by the National Institutes of Health, are gratefully acknowledged. We thank Mat-

452

thias Händel, Arkadiusz Wieczorek, Thomas Ritschel (FSU Jena) and Christian Schröder

453

(University of Stirling) for their assistance in FTIR spectroscopy, SEM, PMF and Mössbauer

454

spectroscopy, respectively. Moreover, we want to thank two anonymous reviewers for their

455

helpful comments.

456



457

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642

643 644

Figure 1: Fe concentrations measured in C. elegans (mainly J3 and J4) after 6 h exposure to

645

K-medium (Control) and ferrihydrite colloids associated with citrate (Fh_citrate) (28 mg Fe l-

646

1

647

comprises bioaccumulated and attached Fe; 8 h post exposure: comprises bioaccumulated Fe;

648

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); 0 h post exposure: comprises bioaccumulated, attached and ingested Fe; 2 h post exposure:


Page 29 of 33


649 650

Figure 2: Response of C. elegans (% inhibition of reproduction compared to control) to syn-

651

thetic and soil effluent FeOx, as well as ionic Fe3+ after 96h of exposure; concentration-

652

response curves were fitted to toxicity data using a sigmoidal logistic model; for abbreviations

653

see Table 1; ECx values are presented in Table 2; for Fh_soil only the undiluted sample

654

(highest concentrations) were tested.



Page 30 of 33

Table 1: Fe oxide (FeOx) properties (values: arithmetic mean  σ) 1)

FeOx

Fh_small

Fh_med

Fh_large

Fh_citrate

Fh_HA

Aka

Goe

2)

SEM4) % in size class (nm diameter *) < 50 50-200 >200

3)

XRD / FTIR

2-line Fh with traces of Hem and Goe

col

2-line Fh with traces of Hem and Goe

col + non-col

2-line Fh with traces of Hem and Goe 2-line-Fh with traces of Goe

2-line-Fh with HA

Aka

Goe

non-col

col

col

col

col

76  0

00

0

71  2

65  2

82  1

70  0

23  1

64  2

0

26  2

34  2

18  1

26  0

11

36  2

100

43

21

00

40

SSA (m2 g-1) 5)

274

Fe (mg l-1)6)

pH6)

3

5.4

7)

not measured

not measured

275

8)

not measured

219

163

8)

9)

6

5.6

17

5.9

11

5.7

20

5.8

28

5.9

11

5.4

Hydrodynamic diameter (dH; nm)

Zeta potential (UE; mV) 12 ± 10

not possible

§

16 ± 12 20 ± 16 16 ± 15

not possible

§

19 ± 14 18 ± 11 18 ± 13

not possible

§

20

5.5

20 ± 12

28

5.6

11

5.4

187 ± 81

-20 ± 12

20

5.5

191 ± 86

-21 ± 17

28

5.5

218 ± 96

-24 ± 15

20

6.2

160 ± 71

-29 ± 18

279

7.3

144 ± 56

-31 ± 13

447

7.5

142 ± 61

-30 ± 20

1

5.3

20 ± 11

5 ± 18 not possible

§

3

5.4

23 ± 11

11

5.5

11

5.6

406 ± 224

-12 ± 16

22

5.8

848 ± 300

23 ± 13

34

6.0

1362 ± 569

22 ± 15

24 ± 11


Page 31 of 33


Table 1 continued Goe_HA

Goe with HA

col

73  3

Fh_soil (BD) rep. Fh_soil (BD) Fh_soil (AD)

24  4

41

not possible$ organo-mineral Fh10)

col + noncol

63  1

37  1

10

34

6.2

417 ± 200

-29 ± 9

112

6.5

329 ± 165

-28 ± 10

168

6.9

305 ± 194

-30 ± 12

not measured

30

7.9

not measured

28

7.9

not measured

15

5.8

not measured

not possible§ 256 ± 134

-17 ± 4 -17 ± 4 -34 ± 5

not measured rep. Fh_soil (AD) 5.8 249 ± 133 -33 ± 7 63  5 37  6 11 15 Fh = ferrihydrite, Goe = goethite, Aka = akaganeite, Hem = hematite, HA = humic acid, BD = before dialysis, AD = after dialysis; rep.: independent replicate 2) XRD = X-ray diffraction; corresponding diffractograms in SI 2 3) FTIR = Fourier-transform infrared spectroscopy; corresponding spectra in SI 3 4) SEM = Scanning electron microscopy; col = colloidal aggregates, non-col = non-colloidal aggregates; corresponding secondary electron images in SI 5 5) SSA = Specific surface area from N2-BET analysis 6) Fe concentrations and pH in FeOx suspensions for low vs. medium vs. high toxicity 7) personal communication: Juliane Braunschweig 8) taken from Bosch et al. 5 9) specified by manufacturer 10) revealed by Mössbauer spectroscopy (SI 4) and FTIR spectroscopy (SI 6) * calculated from aggregate area assuming spherical shape § interferences by non-dispersed aggregates $ interferences by minerals that precipitate from solution owing to drying-induced supersaturation 1)



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Table 2: Low and median effect concentrations (EC20; EC50; ± standard error) for tested compounds (FeOx, Fe3+, paraquat, fluoranthene) calculated for effects on the reproduction of two strains of C. elegans (N2; sod-2) after 96 h of exposure, from dose-response curves (for N2: see also Fig. 2) fitted with a logistic model. EC20 (mg Fe l-1) FeOx1)

EC50 (mg Fe l-1)

N22)

sod-23)

sod/N2

N2

sod-2

sod/N2

6.5 ± 2.4 (AB) 9.4 ± 1.8 (AB)

6.6 ± 1.5 6.6 ± 1.6

1.03 0.70

13.2 ± 2.9 (AB) 17.9 ± 2.1 (B)

13.3 ± 1.9 14.2 ± 1.6

1.01 0.79

Fh_large

15.9 ± 4.0 (BCD)

12.7 ± 2.2

0.80

27.5 ± 2.9 (CD)

21.1 ± 1.6

0.77*

Fh_citrate

22.5 ± 0.33 (C)

11.2 ± 3.1

0.50

31.7 ± 0.21 (D)

20.1 ± 2.7

0.64*

Fh_small Fh_medium

Fh_small + HA

> 212

4)

> 212

4)

-

> 212

a

> 212

1

-

Aka

2.2 ± 1.6 (A)

2.0 ± 8.7

0.90

4.0 ± 3.0 (A)

4.1 ± 4.0

1.02

Goe

23.3 ± 0.022 (D)

23.3 ± 6.0

1.00

29.0 ± 0.022 (C)

34.7 ± 5.4

1.20

129 ± 3.1 (E)

139 ± 0.19

1.07

172 ± 2.8 (E)

143 ± 259

0.83

Fe (citrate)

8.3 ± 0.33

4.1 ± 1.92

0.50

12.1 ± 0.38

8.9 ± 1.7

0.74*

PQ FA

14.1 ± 0.14 0.115

1.0 ± 0.0005 0.097 ± 0.063

0.07* 0.93

19.0 ± 0.12 0.185

1.4 ± 0.0005 0.21 ± 0.12

0.08* 1.20

Goe + HA 3+

1)

Fe oxides (for abbreviations see Table 1); PQ = paraquat (positive control oxidative stress); FA = fluoranthene (negative control oxidative stress) N2 = wild type; capital letters indicate significant differences of EC20/50 between FeOx treatments (two-tailed Z-test) 3) sod-2 = mutant strain hypersensitive to oxidative stress 4) No effect at maximal tested concentration 5) Z-Test not performed, because non-linear model was not significant (ANOVA: p > 0.05) * significant difference of EC20/50 between N2 and sod-2 (one-tailed Z-tests) 2)


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