The toxicity of silver nanoparticles to zebrafish embryos increasesthrough sewage treatment processes

Elke Muth-Kohne • Laura Sonnack • Karsten Schlich • Florian Hischen •

Werner Baumgartner • Kerstin Hund-Rinke • Christoph Schafers •

Martina Fenske

Accepted: 8 August 2013

� Springer Science+Business Media New York 2013

Abstract Silver nanoparticles (AgNPs) are widely believed

to be retained in the sewage sludge during sewage treatment.

The AgNPs and their derivatives, however, re-enter the

environment with the sludge and via the effluent. AgNP were

shown to occur in surface water, while evidence of a potential

toxicity of AgNPs in aquatic organisms is growing. This study

aims to examine the toxicity of AgNPs to the embryos of the

aquatic vertebrate model zebrafish (Danio rerio) before and

after sewage treatment plants (STPs) processes. Embryos

were treated with AgNP (particle size:[90 % \20 nm) and

AgNO3 in ISO water for 48 h and consequently displayed

effects such as delayed development, tail malformations and

edema. For AgNP, the embryos were smaller than the controls

with conspicuously smaller yolk sacs. The corresponding

EC50 values of 48 hours post fertilization (hpf) were deter-

mined as 73 lg/l for AgNO3 and 1.1 mg/l for AgNP. Whole-

mount immunostainings of primary and secondary motor

neurons also revealed secondary neurotoxic effects. A TEM

analysis confirmed uptake of the AgNPs, and the distribu-

tion within the embryo suggested absorption across the skin.

Embryos were also exposed (for 48 h) to effluents of AgNP-

spiked model STP with AgNP influent concentrations of 4 and

16 mg/l. These embryos exhibited the same malformations

than for AgNO3 and AgNPs, but the embryo toxicity of the

sewage treatment effluent was higher (EC50 = 142 lg/l; 48

hpf). On the other hand, control STP effluent spiked with

AgNPs afterwards was less toxic (EC50 = 2.9 mg/l; 48 hpf)

than AgNPs in ISO water. This observation of an increased

fish embryo toxicity of STP effluents with increasing AgNP

influent concentrations identifies the accumulation of AgNP

in the STP as a potential source of effluent toxicity.

Keywords Silver nanoparticles � Silver � Zebrafish

embryo � Sewage treatment processes � Toxicity

Introduction

Silver nitrate has long been known for its antimicrobial

properties (Grier 1983; Russell and Hugo 1994) Nowadays,

it is widely used in a range of consumables and everyday

products, like cosmetics and healing creams, textiles, and

household articles (reviewed by Fabrega et al. 2011;

Nowack et al. 2011) in the form of silver nanoparticles

(AgNPs). The AgNPs enter the aquatic environment by

different pathways, like through the use of cosmetics or

washing of impregnated textiles (Benn and Westerhoff

2008; Geranio et al. 2009). The projected amount of silver

released into the environment for 2010 by wash out effects

from biocidal plastics and cosmetics was claimed to

account for 15 % of the total released silver, which was

calculated to reach 4 lg/l of Ag in sewage plant influents

(Blaser et al. 2008). Whereas the toxic effects of silver in

free ionic form from AgNO3 on microorganisms and

freshwater animals, like fish and invertebrates, are care-

fully investigated (Erickson et al. 1998; Hogstrand and

Wood 1998; Ratte 1999), the examination of the toxic

potential of AgNPs is still in its infancy. Once AgNPs have

E. Muth-Kohne � L. Sonnack � M. Fenske (&)

Fraunhofer Institute for Molecular Biology and Applied Ecology

IME, Forckenbeckstrasse 6, 52074 Aachen, Germany

e-mail: martina.fenske@ime.fraunhofer.de

K. Schlich � K. Hund-Rinke � C. Schafers

Fraunhofer Institute for Molecular Biology and Applied Ecology

IME, Schmallenberg, Germany

F. Hischen � W. Baumgartner

Department of Cellular Neurobionics, Institute of Biology II,

RWTH Aachen University, Aachen, Germany

123

Ecotoxicology

DOI 10.1007/s10646-013-1114-5

entered the wastewater stream, their impact on aquatic

organisms is hard to foresee, as they show chemical and

physical properties different to dissolved silver, and their

effects are not yet fully understood (Fabrega et al. 2011).

Oxygenating conditions in freshwater, on the other hand,

facilitate the dissolution of particulate silver (Liu and Hurt

2010), creating conditions where the toxicity resembles

free silver from AgNO3. Nevertheless, recent studies on

fish provide evidence that a toxic potential may be asso-

ciated with the exposure to AgNP (Laban et al. 2010;

Kwok et al. 2012). Several studies with zebrafish embryos

(e.g. Asharani et al. 2008; Bar-Ilan et al. 2009; Powers

et al. 2011) showed various sublethal and lethal effects,

ranging from spinal cord deformities to reduced hatching

rate and concentration-dependent mortality, depending on

the size, shape, and formation of AgNPs, and on the

exposure time and duration (for review, see Fabrega et al.

2011). Most interestingly was the identification of neuro-

toxicity indicating effects in the zebrafish embryos, as also

described by Powers et al. (2011).

It is estimated that roughly 95 % of AgNPs are removed

from the effluent by the sewage treatment processes and

retained in the sewage sludge (Benn and Westerhoff 2008;

Shafer et al. 1998). However, the percentages of AgNP

removal are only estimates, and recent data of Ag and AgNP

concentrations in sewage treatment plants (STPs) are lack-

ing, as the routine analysis of silver residues was suspended

at the beginning of the 1990s due to the reduction of silver

entry from photo industry. AgNPs still re-enter the aquatic

environment from STPs, either via the effluent or indirectly

from sewage sludge, which is used as fertilizer. Although re-

use of sewage sludge is strictly regulated in Germany

(sewage sludge regulation AbfKlarV, 1992), EU-wide reg-

ulation (guideline 86/278/EWG) is less strict what the

agricultural usage of sewage sludge is concerned.

Even though most of the introduced AgNPs are removed

or bound as water-insoluble salts (Lowry et al. 2012; Kaegi

et al. 2013), the amount of total silver including AgNPs and

of free silver ions still released into the aquatic environ-

ment is unknown and so is the toxic potential associated.

Most of the ionic silver in wastewater and in sewage sludge

reacts with organic or inorganic matter, forming aggregates

and salts of variable solubility and toxicity, like silver

sulfide (Ag2S) or silver chloride (AgCl) (for review, Levard

et al. 2012). The same is postulated for AgNPs since Ag2S-

NPs were identified in sewage sludge of a STP (Kim et al.

2010). Formation of Ag2S (and therefore presumably also

of Ag2S-NPs) can alter the toxic potential of silver, because

in this reduced state, the release of active free Ag ions also

from AgNPs, is inhibited (Choi et al. 2008, 2009; Reinsch

et al. 2012).

A convenient organism to study the impact of AgNPs on

aquatic organisms is the zebrafish (D. rerio) embryo. The

assets of the zebrafish embryo model are favorable for

toxicity assessments in ecotoxicology (Scholz et al. 2008;

Yang et al. 2009), and they have also proven to be bene-

ficial for the testing of nanoparticle toxicity (Asharani et al.

2008; Bar-Ilan et al. 2009; Bilberg et al. 2012; Jovanovic

et al. 2011; Powers et al. 2011).

Here, we describe the effects of AgNO3 and AgNPs

(OECD reference material NM-300K) on zebrafish

embryos, in a 48 h zebrafish embryo toxicity test. Com-

plementary, the damaging effects on primary motor neuron

(PMNs) and secondary motor neuron (SMNs) development

were investigated according to Muth-Kohne et al. (2012),

aimed at evaluating potential neurotoxic effects of AgNPs.

Furthermore, the impact of sewage plant treatment pro-

cesses on AgNP toxicity was studied by testing the efflu-

ents of three model STPs, which were dosed with AgNP

influent concentrations of 0, 4 and 16 mg/l. The primary

aim of this investigation was to extend current knowledge

regarding the toxicity of AgNPs following STP processes

using fish embryos.

Materials and methods

Handling of AgNP and preparation of the silver test

solutions and suspensions

The NM-300K reference AgNP were handled according to

a draft standard operating procedure specifically developed

for this reference material of the European Commission

Joint Research Centre (JRC) (Klein et al. 2011). It is used

in the context of the OECD Working Party on Manufac-

tured Nanomaterials (WPMN) Sponsorship Programme.

NM-300K and the AgNP-free stabilization agent, NM-300

DIS, were obtained from Mercator GmbH, Germany.

The initial suspension of NM-300K contained 10.16 %

of AgNP (w/w) in stabilization agent NM-300 DIS, con-

taining 4 % (w/v) of polyoxyethylene, glycerol, trioleate,

and polyoxyethylene(20)-sorbitan-monolaureate (Tween

20) each. According to the Material Information Sheet

(Version 10, 2011) the AgNPs had an average primary

particle size of 15 nm with a narrow size distribution

(99 %). The release of ions from NM-300K particles into

the matrix under storage conditions was estimated to be

less than 0.01 % (w/w) (Klein et al. 2011). For testing, the

NM-300K suspension (2 ml) was agitated and sonicated in

an ultrasonic bath for 150 (frequency: 40 kHz; USR 9,

Merck Eurolab, Germany) to disperse air bubbles and

AgNP agglomerates prior to the preparation of stock sus-

pensions. A 5 ml suspension—of a concentration of 2 %

(w/w) AgNP was then made up in ISO water (containing

400 lM CaCl2�H2O; 100 lM MgSO4�7H2O; 155 lM

NaHCO3 and 15.5 lM KCl in distilled water, according to

E. Muth-Kohne et al.

123

OECD guideline 203, Annex 2 (OECD 1992)), which was

used as dilution medium for all experiments. For each

experiment, the 2 % stock suspension was agitated and

sonicated for 150 to be used for the preparation of a

working stock solution with a nominal concentration of

200 mg/l [0.02 % (w/w)] total Ag in form of AgNPs. From

this working stock, the test dilutions were prepared in

aerated ISO water, to achieve nominal AgNP concentra-

tions of 0.5, 0.66, 0.87, 1.15, 1.5 mg/l. Corresponding

dilutions were prepared from the stabilization agent with-

out AgNP to test for NP unrelated toxicity.

Accordingly, dilutions of AgNO3 (Sigma Aldrich, Ger-

many) were prepared in aerated ISO water from a stock

solution of 200 mg/l AgNO3 (prepared in ddH2O to avoid

precipitation), to obtain nominal Ag? concentrations (as

AgNO3) of 0.051, 0.063, 0.076, 0.095, 0.121 mg/l. Stock

and test solutions were stored in the dark at room tem-

perature. All test solutions (50 ml in 100 ml screw-cap

glass bottles each) were continually stirred on a magnetic

stirrer at 300 rpm over night to avoid precipitation and to

guarantee a homogenous dispersion. The pH value of the

ISO water and all test solutions/suspensions was kept at

7.2 ± 0.3 (mean ± SD).

Exposure of zebrafish embryos

Wild type zebrafish (D. rerio) originally obtained from

West Aquarium GmbH (Bad Lauterberg, Germany) and

continuously bred for several generations in the Fraunhofer

IME laboratories since, were used for testing. For egg

production, fish were maintained in large 200–300 l tanks

at 27 ± 1 �C on a 14:10-h photoperiod in a temperature-

controlled room. For egg collection, spawning trays were

inserted into the tanks at the beginning of the light period

and left for 1 h.

Exposures were performed in 96-well plates, with one

plate representing one replicate within a test. To account

for the adherence of any test compound to the plastic

surfaces of the polystyrol-microtiter test plates (Greiner

Bio-one, Germany) used for the test, the plates were filled

with 200 ll of the corresponding test solution or ISO water

and incubated overnight at 27 �C to saturate the plastic

surfaces. The remaining test solutions were aerated over-

night and used to replace the solutions in the saturated

plates with 200 ll of the corresponding test solution prior

to the test start. Embryos (at approx. 1 h post fertilization,

hpf) were then individually transferred to the wells (one

embryo/well) and incubated for 48 h at 27 ± 1 �C at a

14:10-h photoperiod. For the AgNP and AgNO3 exposures

in ISO water, each replicate plate contained 24 treated and

12 untreated embryos, as an internal test control. For the

STP effluent exposure, 12 treated and 12 untreated internal

control embryos were used per plate. Each test consisted of

two replicates each for the controls and the treatments and

was repeated three (STP effluent experiments) to five

(AgNP and AgNO3 experiments in ISO water) times.

Exposure time in all experiments was 48 h, with assess-

ments of effects after 24 and 48 h.

Sublethal and lethal morphological effects were evalu-

ated according to Braunbeck and Lammer (2006). The

effective concentration range of AgNP and AgNO3 used in

the experiments were determined previously by range

finding experiments.

Effluent samples of the 4 mg/l AgNP and 16 mg/l AgNP-

dosed model STPs were tested undiluted and serially diluted

with ISO water. A dilution factor of 1.32 was applied to the

samples of the 4 mg/l AgNP dosed model STP and of 1.41

to those of the 16 mg/l AgNP dosed model STP. The

maximum total silver concentrations of the neat and the

diluted effluent samples were calculated from the AgNP

concentration dosed to the influent and thereby reflect the

theoretical maximum total silver concentrations in the

effluents if no silver was retained in the STP. The actual

total silver concentrations in the effluents were determined

only after the experiments (see ‘‘Determination of total sil-

ver concentrations by inductively coupled plasma–optical

emission spectrometry (ICP–OES)’’ section). Considering

the corresponding dilution factors, deduced maximum con-

centrations tested on the embryos were 0.5, 0.66, 0.87, 1.14,

1.5 mg/l of total Ag in case of the 4 mg/l-AgNP STP and

0.25, 0.35, 0.5, 0.71, 1 mg/l of total Ag in case of the 16 mg/

l-AgNP STP. In parallel, AgNP were spiked to effluent of

the AgNP-free model STP and tested as reference controls.

The AgNP concentration used for spiking were 0.5, 0.66,

0.87, 1.15, 1.5, 4, 8, 16 mg/l, to cover the effective con-

centration range. All STP sample dilutions were stirred for at

least 2 h prior to testing to ensure an even dispersion of

particles.

Whole-mount immunofluorescence staining

After the 48 h exposure, surviving AgNP-exposed and

unexposed embryos were manually dechorionated, and

washed 3 times for 50 in ISO water to remove excess AgNPs.

The embryos were then fixed in 4 % (w/v) paraformaldehyde

(Sigma Aldrich, Germany) in phosphate buffered saline

(PBS, Invitrogen, Germany) for 4 h at room temperature,

then rinsed three times for 100 in PBS/0.1 % (v/v) Triton

X-100 (PBST) and stored at 4 �C. Whole-mount immuno-

stainings were performed as described in a book chapter

written by Westerfield (2000) with the following modifica-

tions: Fixed embryos were permeabilized in ice-cold acetone

for 70 at -20 �C, rinsed with distilled water for 50 and washed

three times for 100 with PBST. To prevent nonspecific

binding, the embryos were incubated in blocking solution

(PBST/2 % (v/v) normal goat serum) for 4 h at room

The toxicity of silver nanoparticles

123

temperature at agitation, and then incubated with the primary

antibody (5 lg/ml in blocking solution) overnight at 4 �C.

The embryos were washed five times for 100 with PBST and

subsequently incubated with the corresponding secondary

antibody (1:1,000 dilution) for 4 h at room temperature.

They were washed another five times for 100 with PBST and

mounted on glass slides in glycerin for imaging.

The primary antibodies used were znp1, a mouse mono-

clonal antibody (IgG2a) specific to PMNs, and zn8, a mouse

monoclonal antibody (IgG1) specific to SMNs (Trevarrow

et al. 1990). These were obtained from the Developmental

Hybridoma Bank, University of Iowa, USA. The secondary

antibodies used were DyLight 549-conjugated AffiniPure

Goat Anti-Mouse IgG, specific for Fc subclass 1, and Dy-

Light 649-conjugated AffiniPure Goat Anti-Mouse IgG,

specific for Fc subclass 2a (Jackson ImmunoResearch

Europe). The embryos were microscopically analyzed using

a Leica AF6000 system with a DMI6000 microscope.

Z-stack image series were acquired of each embryo; the

images were processed and analyzed using the ImageJ

software (National Institutes of Health). The Plugin ‘‘Stack

Focuser’’ merges the different focal planes of the z-stacks to

generate full-focus images (with extended depth of field). All

ventral motor neurons (PMN and SMN) in the area above the

yolk sac extension of the embryo were individually exam-

ined for defects (approximately 10 motor neurons per

embryo) and the severity of defects classified according to

previous publications (Carrel et al. 2006; Sylvain et al. 2010;

Muth-Kohne et al. 2012). The proportion of PMNs and

SMNs with no, mild (i.e., delayed development, axons

lacking stereotyped morphology), moderate (i.e., axons with

ectopic branches or innervating neighboring myotomes), or

severe defects (e.g. truncated axons) were determined for

each embryo analysed. Each percentage value represents the

mean (±SEM) proportion of normally developed motor

neurons, and motor neurons with mild, moderate or severe

effects in embryos derived from 5 independent test replica-

tions, with 5–6 individual embryos analysed each.

Transmission electron microscopy (TEM)

TEM of AgNP-exposed and unexposed control zebrafish

embryos was performed; the method is described in more

detail by Asharani et al. (2008). Briefly, treated and untreated

embryos were dechorionated at 48 hpf and anaesthetised on

ice for 100 and immediately fixed in 2.5 % glutaraldehyde/

2 % formaldehyde in PBS at 4 �C for 48 h. The embryos were

post-fixed for 1 h in 1 % osmium tetroxide (Fluka, Switzer-

land) in PBS, washed 3 times for 150 in ddH2O and stained for

1 h with 2 % uranyl acetate solution in ethanol (Merck,

Germany) in a dark environment. Afterwards, the tissue was

gradually dehydrated in ethanol (at 70, 80, 90, 96 and 100 %,

for 150 each) and washed twice for 300 in propylene oxide

(SERVA, Germany), then stored for 16 h in a mixture of

propylene oxide and epoxy resin (Epon; SERVA, Germany).

After washing in Epon twice for 2 h, the samples were

embedded and polymerised for 48 h at 57 �C. Ultra thin

sections were cut with a Reichert OmU3 ultramicrotome (C.

Reichert AG, Austria) and placed on 200 mesh (200 division

bars on 25.4 mm) nickel grids (Plano GmbH, Germany). To

increase the contrast of the tissue compartments, the ultra thin

sections were incubated with 2 % uranylacetate for 200 fol-

lowed by incubation with 0.2 % lead citrate in a CO2-free

atmosphere for 70. After each incubation, the sections were

washed 4–5 times with ddH2O and air-dried for 150. AgNPs

were enhanced by silver adsorption with an Ag-enhancer Kit

(R-Gent SE-EM, Aurion, Netherlands) for 300 at RT in the

dark, following the manufacturers’ instructions. The enhance-

ment of Ag in the embryos with this kit is based on the prin-

ciple of autometallography (Danscher 1991; Danscher and

Montagnese 1994). Here, Ag and enhancer (a reducing agent,

here hydroquinone), both provided with the kit, form com-

plexes in the presence of metal nuclei, i.e., AgNPs. Treated

sections of AgNP-exposed and unexposed control

embryos and the AgNP in ISO water were analysed

using a Zeiss EM 10C transmission electron microscope

(Zeiss, Germany).

Model STPs and preparation of test suspensions

from effluents

According to the OECD test guideline 303a (OECD 2001),

a simulation test for aerobic sewage treatment was per-

formed. For the test, a lab-scale STP (behrotest� Labor-

klaranlage KLD 4N, Germany) with an aeration vessel and

a secondary clarifier was used. The sewage sludge was

obtained from a municipal STP (Ag content\1 mg/kg dry

matter sludge). Three systems were run, one as a control,

two receiving a continuous addition (influent) of 4 mg/l

and of 16 mg/l of AgNP, respectively, for 18 days. The

room temperature was kept at 20–25 �C and the oxygen

level was controlled at a range of 2.0–3.5 mg/l in the

aeration vessel over the whole test period. In compliance

with the OECD 303a guideline, the mean hydraulic

retention time was 6 h, maintained at a continuous flow of

750 ml of a mixture of synthetic sewage, tap water and Ag

stock dispersion mixed within a tube system. The synthetic

sewage contained 160 mg peptone, 110 mg meat extract,

30 mg urea, 28 mg of anhydrous K2HPO4, 7 mg NaCl,

4 mg anhydrous CaCl2 and 2 mg MgSO4�7H2O. The AgNP

stock solutions were prepared 109 concentrated and stored

at 4 �C in a refrigerator. The pH of the sewage sludge in

the aeration vessel was measured continually and adjusted

using anhydrous dipotassium hydrogen phosphate in case

the pH value fell to \7.0.

E. Muth-Kohne et al.

123

For inoculation of the plant simulation units, the sewage

sludge dry matter content was adjusted to 2.5 g/l with tap

water. Freshly obtained activated sludge was adapted to

laboratory conditions for 5 days by continuous feeding

with just synthetic sewage. Before the addition of the

AgNPs, the sludge of the plant units was synchronized by

mixing, and equal volumes were redispensed to the indi-

vidual units for reinoculation. The dry matter content of the

sludge was determined periodically and sludge was

removed and discarded if the dry matter concentration

exceeded 3 g/l, the content of nitrate, nitrite and ammonia

were measured once a week, dissolved organic matter was

measured daily in influent and effluent, and the flow rate

was checked and adjusted if required. Samples for the fish

embryos tests were taken from the effluents of all three

units on day 0, 4, 7, 11, 14 and 18 after the initial AgNP

spiking.

Determination of total silver concentrations

by inductively coupled plasma–optical emission

spectrometry (ICP–OES)

Prior to silver analysis, the STP effluent samples were

digested. All samples containing AgNPs were vortexed for at

least 10, then 5 ml of each were taken and 2 ml of concen-

trated nitric acid (69 %) (‘‘Supra’’ quality, ROTIPURAN�,

Roth, Germany) and 5 ml of hydrochloric acid (30 %)

(‘‘Instra-Analyzed’’, Mallinckrodt Baker, Germany) were

carefully added. The mixtures were incubated at room tem-

perature overnight and afterwards refluxed under heating for

2 h. After cooling, the solutions were slowly and carefully

filled up to a volume of 50 ml with 1 % nitric acid (Klawonn

et al. 2012).

Total silver concentrations in effluents were measured by

ICP–OES using an IRIS Intrepid II ICP–OES (Thermo Sci-

entific, Germany) at wavelengths of 328.068 and 338.289 nm.

At 328.068 nm, no interferences were observed. Therefore,

this wavelength was used for evaluation of measurement

results. A standard containing 1,000 mg Ag/l in nitric acid

2–3 % (Silver ICP Standard, Merck, Darmstadt, Germany)

was used to prepare matrix-adjusted calibration solutions

(same matrix as digested samples). The following calibration

standards were used to calibrate the instrument: blank, 50,

100, 250, 500 and 1,000 lg/l. The limits of determination

(LOD) and quantification (LOQ) were calculated by the

instrument software (LOD: 3 * method standard deviation

from calibration line and LOQ: 10 * method standard devia-

tion from calibration line) to 10.0 and 33.3 lg Ag/l, respec-

tively (ISO 11885 2007).

To determine the accuracy of the applied analytical

method, recalibration samples containing concentrations in

the range of actual samples, and certified reference material

TMDA-70 (certified with 10.9 lg Ag/l, Environment

Canada) were analysed together with the samples. The

recovery rates of Ag (% of deduced maximum Ag) for all

samples analysed are shown in Table 1.

Control effluent samples (no AgNPs added) were ana-

lysed to determine their environmental silver background

level, in order to subtract this background from the measured

silver concentrations in test samples. However, the deter-

mined silver background was below the detection limit.

After the ICP–OES analysis, the measured concentration

values were used to re-calculate the actual total silver

concentrations in the effluent test samples and correct ret-

roactively the deduced nominal Ag concentrations.

Statistical analysis

For the zebrafish embryo tests, concentration-effect curves

with corresponding 95 % confidence intervals (CIs) were

generated. The EC50 and LC50 values for sublethal and

lethal morphological endpoints as well as for PMN and

SMN defects were calculated by applying probit analyses

performed in the ToxRat� Professional program, Version

2.10.

Further statistical analysis was carried out using the

GraphPad Prism software 5.0. All data were tested for

normality and homogeneity of variance. To test for signifi-

cant differences in the percentages of normally developed

motor neurons in the controls compared to normally devel-

oped motor neurons in the treatments, one-way ANOVA

with Dunnett’s method of post hoc testing for multiple

comparisons (*p \ 0.05; **p \ 0.01; ***p \ 0.001) was

applied. To determine significant differences of the calcu-

lated EC50 and LC50 values in the STP experiments, a

Student’s t test (*p \ 0.05; **p \ 0.01; ***p \ 0.001) was

used. In all figures, significant p-values are marked with

asterisks.

The effective concentration values (EC50) for motor

neuron defects were derived from probit analyses of the

concentration-dependent proportional motor neuron defect

data, using ToxRat� Professional, version 2.10. The EC50

values for PMN and SMN defects were normalized against

the corresponding morphological EC50 values after 48 h, to

obtain the two indices, PMNI and SMNI, which provide an

estimate of a developmental neurotoxic potential. SMNI or

PMNI values\1.0 indicate a primary neurotoxic potential.

Results

Morphological effects in zebrafish embryos mediated

by AgNP and AgNO3

Sublethal and lethal morphological effects of AgNP and

AgNO3 were assessed in 24 and 48 h exposed zebrafish

The toxicity of silver nanoparticles

123

embryos and the concentration–response correlations for

cumulative silver-mediated morphological effects of AgNP

and AgNO3 generated (Fig. 1). AgNP as silver source

caused a tenfold lower toxicity in terms of lethality

(Fig. 1a, black squares) and total effects (both, sublethal

and lethal effects; Fig. 1b, black squares) than AgNO3

(Fig. 1a, b, grey triangles). The corresponding 95 % CIs

(dotted lines) showed no overlap between the treatments,

demonstrating a significant difference between AgNP- and

AgNO3-mediated toxicity. The AgNP-stabilization agent

NM-300K DIS alone caused no morphological effects or

mortality at all dilutions used in the exposures (Fig. 1a, b,

blank squares). The calculated mean EC50 values for silver

from AgNP were 1.13 mg/l at 24 hpf and 1.09 mg/l at

48 hpf, whereas the LC50 values were 1.79 mg/l at 24 hpf

and 1.26 mg/l at 48 hpf. The toxicity of silver from AgNO3

was more than ten times lower with an EC50 of 0.09 mg/l at

24 hpf and of 0.07 mg/l at 48 hpf; the LC50s were

0.12 mg/l (24 hpf) and 0.1 mg/l (48 hpf). Longer exposure

of zebrafish embryos (48 hpf compared to 24 hpf) resulted

in a decrease in the EC50 and LC50 values, indicating a

silver-dependent increase in toxicity for both, AgNP and

AgNO3.

Compared to the untreated controls in ISO water, a

characteristic phenotype occurred in AgNP and AgNO3

exposed zebrafish embryos after 48 h of exposure, irre-

spective of the silver source (Fig. 2a). Embryos exposed to

the AgNPs as well as AgNO3 were smaller and had a

smaller yolk sac than control embryos. The development

was delayed in [20 % of the embryos at the highest

exposure concentrations of AgNP and AgNO3. These

effects are highlighted by separate bars in Fig. 2b. The

most conspicuous effect of the treatment was the formation

of a bulky agglomeration that could fill up large propor-

tions of the egg lumen and resembled coagulated material

(see Fig. 2a, both conditions). Minor effects occurring in

AgNP and AgNO3 exposed embryos were the formation of

edema, and malformations of the head and the tail, but

these effects were confined to the two highest test con-

centrations and did affect B20 % of the embryos (data not

shown in Fig. 2b). Overall, the similar phenotype occurring

in both exposures implied a similar mechanism of toxicity

for AgNP as well as AgNO3.

Taking all morphological effects at 48 hpf into account,

a significant increase in affected embryos at a concentra-

tion of 0.87 mg/l AgNP was determined. At the highest

concentration of 1.5 mg/l, 95 % of the embryos were

affected either by morphological aberrations or mortality

(Fig. 2b). The mortality of the embryos contributed to

70 % at 1.5 mg/l of silver in the AgNP dispersion and

increased concentration-dependent (Fig. 2b). Zebrafish

embryos treated with AgNO3 displayed effects increasing

from Ag-concentrations of 0.063 mg/l at 48 hpf. A sig-

nificant number in morphologically affected embryos was

observed from 0.095 mg/l Ag. The highest AgNO3 con-

centration (0.121 mg Ag/l) resulted in 99 % of morpho-

logical aberrations, of which 87.5 % was mortality

(Fig. 2b).

Secondary effect of AgNPs on motor neuron

development

As a neurotoxic effect of AgNPs on zebrafish embryos was

postulated (Powers et al. 2010, 2011), a recently published

indexing system to evaluate the neurodevelopmental

toxicity potential of AgNPs (Muth-Kohne et al. 2012) was

applied to test this assumption. Immunofluorescent stain-

ings revealed indeed a distorted pattern of PMN develop-

ment beginning from the second highest concentration

applied (1.14 mg/l AgNPs). The neurons were heavily

branched and innervated neighboring myotomes (Fig. 3a).

The development of SMNs was delayed and also distorted,

since the axons of the SMNs develop following the axons

of the PMNs (Fig. 3b). Scoring of the severity of the

defects showed a significantly reduced number of normally

developed PMNs at the highest exposure concentration

(Fig. 3c). The delayed development of SMNs of the

zebrafish embryos was considered a mild defect and most

likely linked to the generally delayed development of many

AgNP exposed embryos. As a consequence, a significantly

Table 1 Total silver concentrations (mg/l ± SD) and recovery rates (% of deduced maximum Ag) of Ag standards (Merck, Germany), the

AgNP-dosed model STP influents and the AgNP-dosed model STP effluents

Standard Influent Effluent

Agnom (mg/l) 0.25 0.375 0.5 4 16 4 16

Agcorr (mg/l) 0.251 ± 0.001 0.385 ± 0.0025 0.504 ± 0.005 2.81 ± 0.26 11.5 ± 0.94 0.74 ± 0.22 5.53 ± 1.3

Percentage of nominal (%) 100.5 ± 0.4 102.5 ± 0.6 101 ± 1 70.3 ± 6.5 71.9 ± 5.9 18.4 ± 5.5 34.6 ± 8.1

The table depicts total silver concentrations as nominal (top row), ICP-OES analysis corrected (middle row) and the corresponding recovery rates

(bottom row). Each standard was measured twice. Influents and effluents of STPs were measured at five different sampling days. Note the

increase of total silver proportionally released from the model STP with increasing influent AgNP dosing concentrations (approx. 26 % of total

silver recovery in the effluent at 4.0 mg/l AgNP influent dosing compared to approx. 48 % of total silver recovery in the effluent at 16 mg/l

AgNP influent dosing)

E. Muth-Kohne et al.

123

reduced number of normally developed SMNs were found

at a concentration of 1.14 mg/l AgNPs. The concentration-

effect curve of motor neuron developmental defects

(Fig. 3e) was found to be slightly shifted to the right of the

48 hpf morphological effect curve. This was reflected by

higher EC50 values derived for motor neuron defects (EC50

of 1.5 mg/l AgNPs for PMN defects, and of 1.23 mg/l

AgNPs for SMN defects) compared to the 48 h- cumulative

morphological effect level (1.09 mg/l AgNP). The calcu-

lation of the developmental neurotoxicity indices PMNI

and SMNI (introduced by Muth-Kohne et al. 2012) resulted

in values of 1.33 for the PMNI and of 1.09 for the SMNI,

which would classify AgNPs as neurotoxic, but most likely

only secondary as a consequence of teratogenic effects.

Incorporation of AgNPs into various tissues of zebrafish

embryos

TEM analyses of AgNP-exposed and control zebrafish

embryos at 48 hpf was performed to visualize and confirm

the incorporation of AgNPs into the zebrafish embryos.

TEM images of the AgNP stock solution (Fig. 4a) showed

loosely packed agglomerates of the NM-300K AgNPs with

an average size of approximately 20 nm. Sections of AgNP

exposed embryos revealed an incorporation of silver par-

ticles in the cytoplasm of cells of various embryonic tis-

sues, like the developing gut (Fig. 4b, c), erythrocytes

(Fig. 4f–h) or the cells underlying the retina (Fig. 4l).

Silver particles were also found in extracellular interstitial

spaces (Fig. 4b; arrowhead) or in the lumen of the gut

(Fig. 4b; arrows). An accumulation of silver in particular in

the lumen of the gut and in the erythrocytes was observed.

Corresponding sections of Ag-enhancer-treated control

embryos instead were free of electron dense particles of the

typical size and shape (Fig. 4d, e: developing gut; Fig. 4i–

k: erythrocytes; Fig. 4m: cells underlying the retina),

confirming the identity of silver particles in sections of the

AgNP-exposed fish embryos.

Sewage treatment processes alter the fish embryo

toxicity of AgNPs

Effluent samples from model STPs, spiked with either

4 mg/l or 16 mg/l of AgNPs, were tested on zebrafish

embryos to evaluate the influence of sewage treatment

processes on the toxic potential of the AgNPs, also in

dependency of the influent concentration. The undiluted

effluents of the two STPs with influent concentrations of 4

and 16 mg/l AgNP (compare materials and methods) led

both to 100 % mortality in the embryos. We subsequently

conducted tests with different dilutions of these 4 and

16 mg/l AgNP-dosed effluents in order to investigate dif-

ferent AgNP concentrations in the STP influence in terms

of the toxicity on the zebrafish embryos. The dilutions

tested were chosen to cover the effective range of the

effluents of the two differently treated STPs and corre-

sponded to deduced maximum Ag concentrations ranging

from 0.5 to 1.5 mg/l for the 4 mg/l influent STP simulation

and from 0.25 to 1 mg/l for the 16 mg/l influent simulation

STP. Differences were found between the concentration

Ag from AgNP in ISO Ag from AgNO3 in ISO

Ag [mg/l]

Ag [mg/l]

Stabilization agent w/o Ag (NM-300 DIS) in ISO

leth

al e

ffect

[%]

0.01 0.1 1 100

20

40

60

80

100

50

tota

l effe

ct [%

]

0.01 0.1 1 100

20

40

60

80

100

50

A

B

Fig. 1 Concentration-effect curves of Ag from AgNP in comparison

to Ag from AgNO3 for developmental morphological effects in

zebrafish embryos after 48 hpf exposure, assessed according to the

effect endpoints summarized in Braunbeck and Lammer (2006).

a Percentage of embryos displaying lethal morphological effects after

exposure to AgNP and AgNO3. The red horizontal dashed line

depicts the LC50 value, b Percentage of embryos displaying any lethal

and sublethal effects after exposure to AgNP and AgNO3. The red

horizontal dashed line depicts the EC50 value. Dotted curves indicate

the 95 % CIs. (probit regression; y-axis mean percentage of affected

embryos of 5 independent experiments with 2 replicates of 24

embryos/treatment each; x-axis concentration of total silver as mg/l,

log-scale). Note that based on the nominal Ag concentration, AgNP

(black squares) are 910 less toxic than AgNO3 (grey triangles), in

terms of acute as well as subacute toxicity. The stabilization agent

NM-300 DIS (solvent control; blank squares) exerted no effect (Color

figure online)

The toxicity of silver nanoparticles

123

response of the 4 and the 16 mg/l AgNP STP effluents,

indicating that the AgNP influent concentration has an

impact on the toxicological hazard of AgNPs after sewage

treatment processes. The response curve of the 4 mg/l

AgNP influent simulation was steeper and located between

the curve of the 16 mg/l AgNP influent simulation (Fig. 5,

grey triangles) and the AgNP dispersed in ISO (Fig. 5,

black squares). For clarity reasons, Fig. 5 only presents the

results for the effluents of the 16 mg/l AgNP influent

simulation.

As a reference exposure to the effluent dilutions’ tests,

dispersions of different AgNP concentrations in control

effluent (taken from the STP-simulation without AgNP

dosing) were also tested. In order to achieve comparable

concentration-effect curves, the dilutions of the effluents

were adjusted accordingly (Fig. 5a, b; grey triangles). The

comparison of the concentration-dependent lethal and

sublethal effects (Fig. 5a, b) of these AgNP-spiked effluent

samples (filled circles) with the AgNP-spiked ISO water

(black squares) and the 16 mg/l AgNP-dosed effluent

samples (grey triangles) showed that the curves were

shifted along the x-axis towards lower toxicity. The dif-

ferences in the toxicity were significant, as the 95 % CI of

the curves did not overlap, except for a minor overlap

below the 20 % effect levels of the AgNP in ISO water

curve and AgNPs-spiked reference sample curve (effluent

w/o AgNP in the influent). Same concentrations of AgNP

spiked to (dispersed in) untreated effluent showed a lower

toxicity than in ISO water (Fig. 5a, b; grey dots). The

comparison of the toxicity between the 16 mg/l AgNP

dosed STP dilution samples (grey triangles) and the AgNP

in ISO (black squares) shows therefore the same trend.

ICP–OES analyses confirm increased toxicity

of AgNPs after STP processes

The total silver concentrations of the effluent samples were

determined by ICP–OES. In the effluent of the 4 mg/l AgNP-

dosed STP, the total silver recovery was 18.4 ± 5.5 % of the

AgNP influent concentration, while the recovery in the

effluents of the 16 mg/l AgNP-dosed STP was 34.6 ± 8.1 %

of the AgNP influent concentration (see Table 1). Conse-

quently, the deduced maximum total silver concentrations of

the effluent dilutions tested on the embryos were corrected,

resulting in real concentration values ranging from 0.09 to

0.27 mg/l of total silver for the 4 mg/l AgNP STP dilutions

and from real 0.09 to 0.36 mg/l of total silver for the 16 mg/l

AgNP STP dilutions. The corrected EC50 and LC50 values

00.

051

0.06

30.

076

0.09

50.

121

0

20

40

60

80

100

Ag [mg/l]

Ag from AgNO3 in ISOAg from AgNP in ISO

any effect mortality delayed development smaller with smaller yolk sac

0 0.5

0.66

0.87

1.14 1.

50

20

40

60

80

100

Ag [mg/l]

1,5 mg/L0.87 mg/L 0.121 mg/L0.076 mg/L0 mg/l

w/o AgA

B Ag from AgNP in ISO Ag from AgNO3 in ISO

*

*********

***

with

effe

ct [%

]em

bryo

s

with

effe

ct [%

]em

bryo

s

Fig. 2 Morphological effects of Ag from AgNP and AgNO3 in 48

hpf zebrafish embryos. a Images of control and AgNP- and AgNO3-

treated embryos displaying similar phenotypes with a characteristic

aggregate in the chorion and a smaller habitus with smaller yolk sac,

and retarded development occurred often (magnification of images:

409; scale bars 50 lm). b Concentration-dependent significant

increase in mortality and the characteristic non-lethal effects. ‘‘Any

effects’’ depicts the percentage of embryos showing at least one

adverse effect (y-axis mean percentage of affected embryos of 5

independent experiments with 2 replicates of 24 embryos/treatment;

statistical significance: *p \ 0.05; **p \ 0.01; ***p \ 0.001; one-

way ANOVA with post hoc Dunnett’s test)

E. Muth-Kohne et al.

123

No defects Mild defects Moderate defects Severe defects

0 0.5

0.66

0.87

1.14

0

20

40

60

80

100

1.5

**

% o

f PM

Ns

Ag [mg/l]

C

0 0.5

0.66

0.87

1.14

0

20

40

60

80

100

1.5

*****

% o

f SM

Ns

Ag [mg/l]

D

0.1 1 100

20

40

60

80

100

50

Ag [mg/l]

tota

l effe

ct [%

]

Emorphological effects

effects on SMNs

effects on PMNs

B

cont

rol

0.87

mg/

l1.

5 m

g/l

A

cont

rol

0.87

mg/

l1.

5 m

g/l

zn8

zn8

zn8

znp1

znp1

znp1

Fig. 3 Effects of AgNPs on motor neuron development of zebrafish

embryos at 48 hpf. a Representative fluorescent images of stained

PMNs in control and AgNP-treated embryos. b Representative

fluorescent images of stained SMNs in control and AgNP-treated

embryos. At the highest AgNP-concentration (1.5 mg/l total Ag

nominal), development of SMNs was completely suppressed. c Mean

percentage of PMN defects in AgNP-treated 48 hpf embryos. At

nominal 1.5 mg/l Ag from AgNP, the percentage of normally

developed PMNs was significantly reduced. d Mean percentage of

SMN defects in AgNP-treated 48 hpf embryos. The percentage of

normally developed SMNs was significantly reduced at a

concentration of 1.14 mg/l AgNP. e Concentration-effect curves

(probit regression) of Ag from AgNP after 48 hpf exposure, for

developmental morphological effects (black squares), PMN defect

(grey dots) and SMN defect (grey triangles) in zebrafish embryos.

Rightward shift of the PMN and SMN defects’ curves indicates a

decreased sensitivity of motor neuron damage compared to the

morphological endpoints (magnification of images: 100x; scale

bars 50 lm; statistical significances: *p \ 0.05; **p \ 0.01;

***p \ 0.001; one-way ANOVA with post hoc Dunnett’s test; 5

independent experiments with 5–6 individual embryos)

The toxicity of silver nanoparticles

123

E. Muth-Kohne et al.

123

indicate an increased toxicity of the 4 and the 16 mg/l AgNP

STP effluents compared to values without the concentration

analysis (see Fig. 6 for comparison). The EC50 values (48 h)

were corrected from 0.778 and 0.432 to 0.142 and 0.151 mg/l

and the LC50 values (48 h) from 0.961 and 0.642 to 0.173 and

0.225 mg/l. The concentration correction thereby diminished

the difference in toxicity between the two influent concen-

trations (see Fig. 6, overlaid bars). In contrast, the corrected

Ag exposure concentrations increased the difference between

the toxicity of the 16 mg/l model STP effluents (Fig. 5a, b;

grey triangles) and the AgNPs in ISO water (Fig. 5; black

squares) and the AgNP-spiked control effluents (grey squares)

even further, shifting the corrected response curve (Fig. 5; red

triangles) along the x-axis. These data underscore an increased

toxicity of AgNPs after STP processes.

Discussion

The toxicological impact of silver on aquatic organisms has

widely been recognized (Erickson et al. 1998; Hogstrand

and Wood 1998; Ratte 1999). In recent literature, there is

increasing evidence of biological impacts also for AgNPs

(reviewed in Fabrega et al. 2011), and this was further

corroborated by the present study, which demonstrates a

clear impact on zebrafish embryo development (Figs. 1, 2).

Nevertheless, they are often regarded as ecologically

harmless, as the silver particles are believed to be mostly

retained by sewage treatment processes with no critical

amounts entering the aquatic environment (Benn and

Westerhoff 2008; Blaser et al. 2008; Burkhardt et al. 2010;

Warila et al. 2001). However, the environmental fate of

A

Ag from AgNP in ISO

Ag from effluent (influx 16 mg/l AgNP); corr.

Ag from AgNP in effluent (w/o influx)

Ag from effluent (influx 16 mg/l AgNP)

B

Ag [mg/l]0.01 0.1 1 10 1000

20

40

60

80

100

50

tota

l effe

ct [%

]

Ag [mg/l]0.01 0.1 1 10 1000

20

40

60

80

100

50

leth

al e

ffect

[%]

Fig. 5 Influence of STP processes on AgNP-mediated toxicity in

zebrafish embryos. a Concentration-effect curves of Ag from AgNP

for developmental morphological effects in zebrafish embryos after

48 hpf exposure, assessed according to the effect endpoints summa-

rized in Braunbeck and Lammer 2006. The embryos were exposed to

AgNPs dispersed in ISO (black squares), to STP control effluent

spiked with AgNPs (w/o AgNPs in the influx; grey dots) and to

effluent of a STP dosed with nominal 16 mg/l of total Ag from AgNP

in the influent (grey triangles); the same curve with corrected total

silver concentrations is depicted in red (red triangles). a Percentage of

embryos displaying lethal morphological effects after exposure to

AgNPs. The red horizontal dashed line depicts the LC50 value (48

hpf). b Percentage of embryos displaying any lethal and sublethal

effects after exposure to AgNPs. The red horizontal dashed line

depicts the EC50 value (48 hpf). Dotted curves indicate the 95 % CIs

(probit regression; y-axis mean percentage of affected embryos of 4 or

5 (AgNPs dispersed in ISO) independent experiments with 2

replicates of 12 or 24 (AgNPs dispersed in ISO) embryos/treatment

each; x-axis concentration of total silver as mg/l, log-scale). Note that

the curves for the STP effluents (triangles) are shifted to the left along

the x-axis, indicating a significant increase in toxicity compared to the

AgNPs in ISO (black squares) and spiked to control effluent (grey

squares) (Color figure online)

Fig. 4 TEM images of the AgNPs in ISO and of AgNP-exposed (b,

c, f, g, h, l) and unexposed (d, e, i, j, k, m) 48 hpf zebrafish embryos.

All sections except for a were treated with an Ag-enhancer kit to

indirectly distinguish incorporated AgNPs (according to ‘‘Materials

and methods’’ section). a AgNP stock suspension adsorbed to the

polymer film of a TEM-grid. AgNPs were of an average size of

approximately 20 nm. b Transversal section of the developing gut.

Large aggregates of silver can be seen in the lumen of the gut

(indicated by arrows) and in the interstitium (arrowhead). The box-

framed detail was magnified and is displayed in c, showing silver

particles within the cells lining the lumen of the gut (indicated by

arrows). d Corresponding tissue of an unexposed embryo (transversal

section of the developing gut, caudal to b. e Box-framed mucosal cell

in d, magnified, showing no evidence of AgNPs. f Cross-section of a

blood vessel near the yolk sack of an exposed embryo, showing

several erythrocytes. g, h Box-framed areas in f at higher magnifi-

cation, showing silver particles within the erythrocytes (indicated by

arrows). i Corresponding section of unexposed embryo, j, k Box-

framed erythrocytes in i, magnified, showing no signs of silver

particle presence. l Silver particles within cells underlying the retina.

Particles are seen throughout the cytoplasm (indicated by arrows).

m Corresponding tissue of an unexposed control embryo, again free

of electron dense particles of the typical size and shape of silver

b

The toxicity of silver nanoparticles

123

AgNPs undergoing sewage treatment processes is very

complex (Potera 2010) and will not be understood by

looking at the behaviour of the nano silver particles only.

Our study now demonstrates the effects of AgNPs (NM-

300K, average size \20 nm) on the development of

zebrafish embryos, highlighting specifically the influence

of sewage treatment processes on the toxicity. We assessed

concentration-dependent morphological effects in the

embryos after exposure to AgNPs or AgNO3, and com-

pared these effects and the thresholds to the toxicity of

effluents of AgNP dosed model STPs and AgNP-spiked

control effluents. Overall, the exposed embryos displayed a

characteristic phenotype irrespective of the silver species.

AgNPs as well as AgNO3 similarly caused development

retardation and smaller than normal embryos were fre-

quently observed at higher concentrations as well as the

formation of conspicuous agglomerations in the chorion

(compare Fig. 2a). Such phenotypes after AgNP exposure

of embryos were reported previously for zebrafish (Asha-

rani et al. 2008; Bar-Ilan et al. 2009) and also for the

Japanese medaka (Oryzias latipes) (Wu et al. 2010).

Reports of additional malformations like abnormal body

axes and pericardial edema and of cardiac arrhythmia at

48 hpf after AgNPs exposure (Asharani et al. 2008; Powers

et al. 2011) could, however, not be confirmed by our data.

In the present study, similar effects were observed only in

very few embryos and to a far lesser extent than the pre-

viously mentioned prevailing effects (Fig. 2), or they

occurred as secondary effects subsequent to the delay in

development. Differences in phenotype and effect severity

can be related to size, the coating, or the stabilization agent

of the nanoparticles used, even when the exposure condi-

tions are similar (El Badawy et al. 2011; Johnston et al.

2010; Powers et al. 2011). This could have also contributed

to the phenotypic differences to our study, as no coating

was used and the AgNP capping (stabilization agent NM-

300K DIS) was different and exerted no effect on the

zebrafish embryos (compare Fig. 1, blank squares).

The previously postulated hypothesis that AgNPs have a

neurotoxic potential (Powers et al. 2010, 2011) could not be

confirmed by our results. Although conspicuous impair-

ments of motor neuron development in the zebrafish

embryos were observed, a significant decrease in normally

developed motor neurons was only confirmed for concen-

trations above the morphological effect threshold and EC50

value (compare Fig. 3). The calculation of the PMNI and

SMNI (Muth-Kohne et al. 2012) therefore resulted in values

[1, which implied that the motoneuronal defects were only a

consequence of a general teratogenicity and a delay in the

overall development. However, our results do not necessarily

refute the findings of Powers et al. (2010, 2011), as they

examined different, citrate-coated AgNPs and assessed

mainly neurobehavioural effects at a later developmental

stage of zebrafish, i.e., at 5 dpf.

Previous studies had demonstrated that AgNPs penetrate

the chorion (Lee et al. 2007), thus a successful uptake by

the embryo without dechorionation was assumed in our

study. However, to confirm this assumption, exposed and

untreated control embryos were analysed by TEM (Fig. 4).

The analysis of sections of exposed zebrafish embryos

suggests a distribution of particulate matter in a variety of

tissues and in particular an accumulation in erythrocytes

and the gut. Equally treated but AgNP-unexposed embryos

were free of particulate matter (Fig. 4), which gave us the

confidence that these particles were indeed the AgNPs. The

random distribution of the AgNPs across varied tissues of

the embryo suggested a primary absorption of the AgNPs

through the skin or by endocytosis during early stages of

development, which was also assumed by Asharani et al.

(2008). Uptake via the gills is unlikely, as the gills were not

yet developed at this time point of embryogenesis.

In principle, the results of this study corroborate existing

evidence that the AgNP-related effects on the development

of zebrafish embryos might be mediated by silver ion

toxicity (Xiu et al. 2012). The observed retarded develop-

ment, on the other hand, can presumably be attributed to

reduced oxygen supply, as hypothesised previously (Kuster

Ag from AgNP in ISOAg from AgNO3 in ISOAg from AgNP in effluent (w/o influx)Ag from effluent ( AgNP influx 16 mg/l; AgNP influx 4 mg/l) Ag from effluent ( AgNP influx 16 mg/l; AgNP influx 4 mg/l); corr.

0.01

0.1

1

10

100

EC 24hpf EC 48hpf LC 24hpf LC 48hpf

*** ** ** *** *** *** ***

** * *ns

Ag

[mg/

l]

Fig. 6 Comparison of EC50 and LC50 values (24hpf and 48 hpf,

morphology) of the AgNP and AgNO3 zebrafish embryo exposures.

White (AgNP in ISO), light grey (AgNO3 in ISO), dark grey (STP

control effluent spiked with AgNP) and black/grey (STP effluent

dosed with 16 mg/l AgNP and 4 mg/l AgNP in influent, respectively)

coloured bars are depicted as nominal total Ag concentrations; the

red/light red bars represent the corrected concentration values,

correction factor 0.35 and 0.18 (for STP effluent dosed with 16 mg/l

AgNP and 4 mg/l AgNP, respectively); (Statistical significances:

*p \ 0.05; **p \ 0.01; ***p \ 0.001; Student’s t test; mean data of

3–5 independent experiments with 2 replicates of 12 and 24 embryos,

respectively, each). Note that concentration correction of the EC50

and LC50 values of the AgNP-dosed STP effluents showed that the

actual toxicity of these samples was higher than originally suggested

by the calculated total Ag concentrations. A significantly higher toxic

potential of AgNP after STP processes is indicated (Color figure

online)

E. Muth-Kohne et al.

123

and Altenburger 2008; Strecker et al. 2011). The quality of

the observed effects was highly similar between AgNP and

AgNO3. Although the toxicity of the AgNPs was ten times

lower compared to AgNO3 (Fig. 2), this lower toxicity of

the AgNP is not surprising and rational, given the slow

dissociation and release of dissolved Ag? ions described

for nano silver (Bilberg et al. 2012; Laban et al. 2010),

which would have contributed only to a small fraction of

the total silver measured in the AgNP samples. Most

interestingly, however, was our discovery that the toxicity

of the effluents was significantly higher than for the AgNPs

dispersed in ISO or when spiked to control effluent.

The chemical analysis of the effluents of the model STPs

recovered only 18 % of total nominal silver originally

dosed to the 4 mg/l AgNP STP, and 35 % of the total silver

concentration was released from the 16 mg/l AgNP-dosed

STP. These silver recovery rates of\50 % would suggest a

clear reduction in embryo toxicity. Instead, a decrease in

embryo toxicity was reflected, shown, for instance, by the

significant, almost tenfold decrease in the EC50 values

obtained from AgNP-dosed STP effluent exposures and

AgNP dispersions in ISO water (see Table 1 for compari-

son). The corrected EC50 (48 hpf) values of the 4 and

16 mg/l AgNP effluents (0.142 and 0.151 mg/l, respec-

tively) rather resembled the toxicity of AgNO3 (EC50 of

0.073 mg/l at 48 hpf). We hypothesise as one possible

factor contributing to these findings the exhaustion of

sulphide and chloride in the sewage sludge. As said earlier,

silver reacts with reducing agents like sulphide or chloride

in the sewage sludge to form stable, non-soluble aggregates

like Ag2S and AgCl, and the transformation rate can be

close to 90 % (Kaegi et al. 2011; for review, Levard et al.

2012). At higher concentrations of AgNPs (5 mg/l), an

exhaustion of sulphide was reported for a STP simulation,

which reduced the transformation of AgNPs to \50 %

(Kaegi et al. 2011). Given the measured total silver

recovery rates in our study, combined with the increase in

toxicity from before and after STP treatment, this

hypothesis is clearly supported by our data. A definite

confirmation, however, would require a comprehensive

physico-chemical characterization of the sludge and efflu-

ent samples.

Generally, the findings of this study in terms of fish

embryo toxicity, substantiate widely the prevailing evi-

dence of a predominantly free silver ion-driven toxicity of

AgNP in fish embryos (Bar-Ilan et al. 2009; Bilberg et al.

2012; Chen and Schluesener 2008; Xiu et al. 2012). Sup-

porting evidence is provided by the coherent morphologi-

cal effects and toxicity thresholds of AgNO3 and AgNP,

together with significantly lower toxicities induced by

AgNP dispersed in ISO water or control effluent. Addi-

tionally, the reduction in particle size in the course of the

STP processes may have also influenced the embryo

toxicity of the effluents, as the toxicity of AgNPs and other

nanoparticles was shown to also depend on the particle size

(Baker et al. 2005; Bar-Ilan et al. 2009; Nino-Martınez

et al. 2008; Oberdorster et al. 2005). Smaller sizes impli-

cate higher surface-to-volume ratios, therefore facilitating

the release of silver ions (Bar-Ilan et al. 2009). Yet, the size

distribution was not measured in this study, so it remains

speculation whether the reduction of the AgNP size was a

contributing factor to the toxicity increase during STP

processes.

In conclusion, the results of this study demonstrate that

under experimental conditions AgNPs can cause major

developmental impairments in zebrafish embryos already

at concentrations below 1 mg/l. Moreover, through STP

processes this toxicity can even increase significantly

reaching a toxicity range comparable to AgNO3. These

findings highlight the risk of extensive use of AgNPs,

which should be considered more specifically, as the risk

associated with the entry of AgNPs to the aquatic envi-

ronment is difficult to predict realistically due to fact that

their properties and behaviour in media like sewage sludge

are not yet fully understood.

Acknowledgments This work was partially supported by the Fra-

unhofer Gesellschaft (FhG) internal programs under Grant No. Attract

692093. We thank Dr. Thorsten Klawonn for performing the ICP–

OES analytics.

Conflict of interest The authors declare that they have no conflict

of interest.

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