Uptake routes and toxicokinetics of silver nanoparticles and silver ions in the earthworm Lumbricus rubellus.

TL;DR: The importance of oral exposure questions the application of current metal bioavailability models, which implicitly consider that the dominant route of exposure is via the soil solution, for bioavailability assessment and modeling of metal-based NPs.

Abstract: Current bioavailability models, such as the free ion activity model and biotic ligand model, explicitly consider that metal exposure will be mainly to the dissolved metal in ionic form. With the rise of nanotechnology products and the increasing release of metal-based nanoparticles (NPs) to the environment, such models may increasingly be applied to support risk assessment. It is not immediately clear, however, whether the assumption of metal ion exposure will be relevant for NPs. Using an established approach of oral gluing, a toxicokinetics study was conducted to investigate the routes of silver nanoparticles (AgNPs) and Ag+ ion uptake in the soil-dwelling earthworm Lumbricus rubellus. The results indicated that a significant part of the Ag uptake in the earthworms is through oral/gut uptake for both Ag+ ions and NPs. Thus, sealing the mouth reduced Ag uptake by between 40% and 75%. An X-ray analysis of the internal distribution of Ag in transverse sections confirmed the presence of increased Ag concentrations in exposed earthworm tissues. For the AgNPs but not the Ag+ ions, high concentrations were associated with the gut wall, liver-like chloragogenous tissue, and nephridia, which suggest a pathway for AgNP uptake, detoxification, and excretion via these organs. Overall, the results indicate that Ag in the ionic and NP forms is assimilated and internally distributed in earthworms and that this uptake occurs predominantly via the gut epithelium and less so via the body wall. The importance of oral exposure questions the application of current metal bioavailability models, which implicitly consider that the dominant route of exposure is via the soil solution, for bioavailability assessment and modeling of metal-based NPs.

Summary

INTRODUCTION

• The rapid increase of nanotechnology can be expected to result in increased rates at which engineered nanoparticles (NPs) are entering the environment.
• The nature of some common nanotechnology products, including cosmetics, textiles and personal care products, means that NPs can be expected to enter wastewater streams, where within sewage systems they may sediment into the sludge material.
• Once in the environment, there is the potential for metal-based NPs or metal ions, that are derived following their solubilisation, to come into contact with organisms and hence to be accumulated [4] [5] [6] [7] .
• The prevailing ecotoxicology paradigm states that for effects to occur it is necessary for the material to be taken up into the body, and ultimately reach a target site.
• For organisms that live in soils, NP exposure can be expected to occur through three main routes.

Acc e p ted P r e p r i nt

• All rights reserved al. [10] that used surgical glue to inhibit soil ingestion.
• After a further mixing, all soils were maintained for an initial period of one week to allow for the initial binding and interactions of the added Ag NPs and ions with soil solid phase and pore water components.
• Thus after 168 h, Ag tissue concentrations in sealed earthworms were between 40-75% of those in unsealed individuals across both Ag forms and exposure levels .
• The identification of the gut as the dominant route of uptake of Ag as ion and NPs has a number of specific implications for the way these chemicals can be handled within risk assessment.

Material supply and characterisation

• Uptake studies were conducted with two chemical forms of silver, Ag NPs and Ag + ions derived from AgNO 3 .
• The Ag NPs used for the study were obtained from NanoTrade Ltd (Prague, Czech Republic).
• For initial material characterisation, dispersions of the supplied material (1 mg mL -1 ) were prepared in distilled water for analysis of particle morphology and size distribution analysis using transmission electron microscopy (TEM).
• The instrument used was a JEOL 2010 analytical TEM incorporating a LaB6 electron gun operated between 80 and 200Kv and equipped with an Oxford Instruments LZ5 windowless energy dispersive X-ray spectrometer.
• The Ag nitrate salt (AgNO 3 , 99% purity) used was purchased from BHD Chemicals (Poole, UK) as a white crystalline powder.

Soil selection and spiking

• The soil used for all Ag uptake kinetic studies was standardised LUFA 2.2 loam sand (LUFA-Speyer, Germany) [24] .
• The soil was initially screened through a 2 mm mesh to remove any large coarse material and to break up larger aggregates.
• The batch was then sub-divided into sufficient aliquots for each control, Ag NP spiked and Ag + ion treatment replicate.
• Previous work conducted to assess the toxicity of freshly spiked pristine.

Uptake bioassay and tissue Ag analysis

• All earthworms used were morphologically determined as Lumbricus rubellus (supplied by Lasebo BV, Nijkerkerveen, The Netherlands).
• This species was chosen because it is a widely distributed epigeic earthworm species in agricultural soils where it may come into contact with Ag in NP and ionic forms added through routes such as sewage sludge.
• Further, L. rubellus has also been found to be suitable for oral gluing studies [10] .
• All earthworms were initially maintained in a stock culture on a medium consisting of 1:1:1 mix of composted bark : Spagnum peat : loam soil and supplied ad libitum with a combination of horse manure collected from animals grazing uncontaminated pasture, and that had not undergone any recent medication and additional vegetable.

µX-ray fluorescence mapping

• Bio-imaging was performed at the Diamond Light Source (UK) using the I18 beamline.
• Sections were mounted on Ultralene® window film 4 µm-thick (SPEX SamplePrep, Metuchen, NJ) stretched across a hole in a plastic slide in order to minimize the Si signal associated with glass substrates.
• Slides were inserted into the standard I18 sample holder, and imaged 'externally' under brightfield conditions for orientation purposes.
• X-ray fluorescence (XRF) data was collected using a Si(111) double crystal monochromator and the Kirkpatrick-Baez focusing mirrors, which provided a 3 µm spot size, were also used to remove harmonic contamination.
• However this provides a relatively insensitive signal as the Ag L(III) edge has a low fluorescence yield [28] .

Data handling and statistical analysis

• Uptake and elimination rate constants were estimated by applying a one-compartment first order kinetics model to the data for the uptake phase, with a constant start value C 0 .
• This model has been widely used in the ecotoxicological literature as a means to assess uptake and elimination kinetics.
• Two sets of values were calculated for the uptake rate, k 1-T was calculated using total measured Ag concentration and k 1-pw using silver concentration in the soil pore water.
• Equations were fitted to the replicate experimental data using least squared regression fitting in SPSS 17.1.
• Significant differences between uptake rates for sealed and unsealed earthworms were compared using a generalised likelihood ratio test [30] .

Material characterization and concentration validation

• The TEM analysis of the supplied nanopowder indicated the presence of primary particles in the 50-80 nm range, with a smaller proportion of smaller 10-30 nm particles.
• These primary particles had often formed into loose agglomerates.
• These were not, however, stable structures as they were readily disintegrated by exposure to the TEM electron beam during analysis.
• The average measured Ag concentrations in replicate soil samples from each treatment were > 85% of nominal values confirming the general efficiency of the spiking procedure (Table 1 ).
• Ag concentrations in pore water samples were ten times higher for the soil spiked with ionic.

Ag uptake in sealed and unsealed earthworms

• During the experiments, the burrowing capacity of sealed and unsealed earthworms was similar and they all disappeared into the soil.
• No obvious visual differences in activity and locomotion were found, although initially the sealed earthworms needed approximately 15 min to begin burrowing.
• Hence the extent of dermal contact with the soils was not affected, when oral exposure was prevented.
• No effect on survival was observed during the 96-h exposure period.
• Ag concentrations in earthworms exposed to the unspiked soil were low (average Ag concentration was 0.02 µg g -1 dry weight, d.w.) over the duration of the exposure.

DISCUSSION

• The successful application of models such as the FIAM and BLM to explain the effects of variations in media properties on metal toxicity [15] [16] [17] has pointed to the validity of the assumptions of these models that relate to the concentration of the ionic form in contact with external surfaces (epidermis, gill).
• Attempts to apply models such as the BLM that assume dermal interactions with the dissolved metal to explain observed metal uptake and toxicity in soils dwelling organisms have generally been informative [15, 16] .
• So far, the application of the FIAM and BLM to explain the effects of metals on soil organisms has not extended to studies with Ag.
• For geophagous groups such as earthworms, the gut-lining, like the skin, will be in near continuous contact with the soil medium.
• Even if gut exposure is actually an important site of uptake, deviations of accumulation rates from those derived from only epidermal exposure will only occur if gut physiology has a substantive influence on the rates of metal ion association with uptake sites or alternatively if the frequency of uptake sites in gut tissues exceeds that of the epidermis.

Figures

Table 2. Uptake and elimination rate constants (related to total (k1-T and k2-T) and soil pore water (k1-pw) silver concentrations) estimated from one-compartment model fits of a time series of total body Ag concentrations measured in the earthworm Lumbricus rubellus exposed to Ag+ ions and Ag NPs in LUFA 2.2 loam sand soil. The 95% confidence intervals are shown in brackets. Coefficient of determination (r2) describes variance in earthworms body concentrations of Ag explained by the fitted model.

Table 1. Nominal, measured and pore water concentrations of Ag in soils sampled at the start of the earthworm uptake experiment with ionic Ag and Ag NPs. Mean values ± standard deviation (SD) are shown.

Full text

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Diez-Ortiz, Maria; Lahive, Elma; Kille, Peter; Powell, Kate; Morgan, A. John;
Jurkschat, Kerstin; Van Gestel, Cornelis A.M.; Mosselmans, J. Fred W.;
Svendsen, Claus; Spurgeon, David J. 2015. Uptake routes and
toxicokinetics of silver nanoparticles and silver ions in the earthworm
Lumbricus rubellus. Environmental Toxicology and Chemistry, 34 (10).
2263-2270. 10.1002/etc.3036
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Environmental Toxicology Environmental Toxicology and Chemistry
DOI 10.1002/etc.3036
UPTAKE ROUTES AND TOXICOKINETICS OF SILVER NANOPARTICLES AND
SILVER IONS IN THE EARTHWORM LUMBRICUS RUBELLUS
Running title: Silver uptake in earthworms occurs mainly via oral exposure
MARIA DIEZ-ORTIZ,*† ELMA LAHIVE,† PETER KILLE,‡ KATE POWELL,‡ A. JOHN MORGAN,‡ KERSTIN
JURKSCHAT,§ CORNELIS A.M. VAN GESTEL,|| J. FRED W. MOSSELMANS,# CLAUS SVENDSEN,† and
DAVID J. SPURGEON†
†
Centre for Ecology and Hydrology, Crowmarsh Gifford, Wallingford, Oxfordshire, United
Kingdom
‡
Cardiff School of Biosciences, University of Cardiff, Cardiff, Wales, United Kingdom
§Department of Materials, Oxford University, Yarnton, Oxfordshire, United Kingdom
||
Department of Ecological Science, Faculty of Earth and Life Sciences, VU University,
Amsterdam, The Netherlands
#Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, United
Kingdom
* Address correspondence to mdiez@leitat.org
This article is protected by copyright. All rights reserved
Submitted 16 June 2014; Returned for Revision 14 August 2014; Accepted 24 April 2015

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This article is protected by copyright. All rights reserved
Abstract: Current bioavailability models, such as the free ion activity model and biotic ligand model,
explicitly consider that metal exposure will be mainly to the dissolved metal in ionic form. With the rise
of nanotechnology products and the increasing release of metal-based nanoparticles (NPs) to the
environment, such models may increasingly be applied to support risk assessment. However, it is not
immediately clear whether the assumption of metal ion exposure will be relevant for NPs. Here using
an established approach of oral gluing we have conducted a toxicokinetics study to investigate the
routes of Ag NP and Ag
+
ion uptake in the soil dwelling earthworm Lumbricus rubellus. Results
indicated a significant part of the Ag uptake in the earthworms is through oral/gut uptake for both Ag
+
ions and NPs. Thus, sealing the mouth reduced Ag uptake by between 40-75%. An X-ray analysis of
the internal distribution of Ag in transverse sections confirmed the presence of increased Ag
concentrations in exposed earthworm tissues. For the Ag NPs but not the Ag
+
ions, high concentrations
were associated with the gut wall, liver-like chloragogenous tissue and nephridia, which suggest a
pathway for Ag NP uptake, detoxification and excretion via these organs. Overall our results indicate
that Ag in ionic and NP form is assimilated and internally distributed in earthworms and that this uptake
occurs predominantly via the gut epithelium and less so via the body wall. The importance of oral
exposure questions the application of current metal bioavailability models, which implicitly consider
that the dominant route of exposure is via the soil solution, for bioavailability assessment and modelling
of metal-based NPs. This article is protected by copyright. All rights reserved
Keywords: Silver, Nanoparticles, Exposure route, Uptake, XANES

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INTRODUCTION
The rapid increase of nanotechnology can be expected to result in increased rates at which
engineered nanoparticles (NPs) are entering the environment. The nature of some common
nanotechnology products, including cosmetics, textiles and personal care products, means that NPs can
be expected to enter wastewater streams, where within sewage systems they may sediment into the
sludge material. The deposition of this waste to land provides a route by which these released NPs may
enter into soil ecosystems [1-3]. Once in the environment, there is the potential for metal-based NPs or
metal ions, that are derived following their solubilisation, to come into contact with organisms and
hence to be accumulated [4-7].
The prevailing ecotoxicology paradigm states that for effects to occur it is necessary for the
material to be taken up into the body, and ultimately reach a target site. For conventional chemicals, the
concepts of toxicokinetics and toxicodynamics are well established as a coherent framework that links
exposure to toxic effects [8]. There is reason to expect that this paradigm will be relevant for NPs,
although some debate remains concerning the extent to which toxicity may be dependent on the
biological interactions resulting once NPs enter tissues and cells. Hence to understand the effects of
NPs it is important to understand key aspects governing uptake into exposed organisms under realistic
conditions.
For organisms that live in soils, NP exposure can be expected to occur through three main
routes. Exposure through air is relevant only for more volatile chemicals and hence for NPs under
normal soil moisture conditions is unlikely to be important. For the remaining two routes, namely
exposure through contact with and transfer across the skin (dermal) and ingestion and transfer across
the gut epithelium (oral), previous studies with conventional chemicals have generally suggested
dermal exposure as the dominant route. This includes pesticides and non-polar organic chemicals in
studies with woodlice and earthworms [9] and for Cd and Zn metal ions in a classic study by Vijver et

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al. [10] that used surgical glue to inhibit soil ingestion. The latter approach allowed for separate
analysis of dermal uptake with and without the additional inputs derived from ingestion and is adopted
here.
Indications of dermal contact as the dominant route of exposure have underpinned the
development of models, such as the free ion activity model (FIAM) [1] and later the biotic ligand model
(BLM) [12] and related terrestrial bioavailability methods, that link soil solution chemistry and metal
speciation modelling, to passive absorption and surface ligand binding on biological membranes,
notably epidermis and gill and thereafter ultimately to toxic effects [11-14]. The successful application
of these models to explain the effects of variations in media properties on metal toxicity [15-17] has
pointed to the validity of the assumptions inherent in these models, with exposure mainly through
external body surfaces. However, although many studies have used the FIAM and BLM to explain
metal bioavailability and toxicity, not all research has necessarily supported this conjecture regarding
exposure routes. For example, the results of Cain et al. [18] suggested that free ion concentrations
accounted for less than 5% of Cd and Cu accumulation in mayflies in aqueous exposure. While the
identification of a dependence of uptake on gut physiology and microbiome composition [19,20] also
indicates dietary uptake as an important exposure route [21].
Previous studies of soil invertebrate exposure to metal-based NPs have established that these
materials can enter into the tissues either as intact particles or following dissociation to ions [6,22,23].
In cases where the uptake of intact NPs is suggested it is, however, currently unclear how these
materials enter tissues. This uncertainty currently places limits on the development of a modelling
framework that can link NP exposure to uptake and effects, such as for example whether assumption of
dermal uptake are valid. To specifically assess the importance of different potential exposure routes of
NP uptake, we conducted a study to assess the toxicokinetic patterns of Ag uptake in earthworms
exposed to both ionic and NP forms of Ag in soil, using the same oral sealing approaches as used by
Vijver et al [10] to quantify the comparative contributions of both the dermal and oral exposure route.

Frequently asked questions

Q1. What are the contributions in "Uptake routes and toxicokinetics of silver nanoparticles and silver ions in the earthworm lumbricus rubellus running title: silver uptake in earthworms occurs mainly via oral exposure maria diez-ortiz,*† elma lahive,† peter kille,‡ kate powell,‡ a. john morgan,‡ kerstin" ?

Vijver et al. this paper conducted a study to assess the importance of different potential routes of nanoparticles uptake, using Ag uptake and NP uptake. 

Q2. What are the future works mentioned in the paper "Uptake routes and toxicokinetics of silver nanoparticles and silver ions in the earthworm lumbricus rubellus running title: silver uptake in earthworms occurs mainly via oral exposure maria diez-ortiz,*† elma lahive,† peter kille,‡ kate powell,‡ a. john morgan,‡ kerstin" ?

Their studies on the relevance of different exposure routes for Ag ions and Ag NP uptake by earthworms are informative for the validity of such future application. Such intake via the gut provides the potential for exposure that may be additional to that resulting from dermal contact alone [ 9 ]. In the latter seminal study, gluing was suggested as a suitable approach for oral sealing, as it allowed normal burrowing and behaviour in the absence of feeding. This suggestion is confirmed by observations made in the present study. 

Q3. What is the current focus in metal and NP ecotoxicological research?

A current focus in metal and NP ecotoxicological research is to understand how environmental conditions such as pH and organic matter, and also for NPs relevant processes such as aggregation and dissolution, are related to toxicity. 

Q4. How long did the earthworms remain in the filter paper?

The earthworms were then kept individually for 36 h in Petri dishes lined with a piece of moistened filter paper to allow them to void their gut content. 

Q5. How long did the soils remain in the test?

After a further mixing, all soils were maintained for an initial period of one week to allow for the initial binding and interactions of the added Ag NPs and ions with soil solid phase and pore water components. 

Q6. How many earthworms were used for the experiment?

A total of 44 sealed and 44 unsealed earthworms were used for each of these treatments (2 x NP; 2 x ionic; 1 x reference) to provide a sufficient number of individuals for collection of 4 replicate earthworms for each of the 10 exposure times used for the uptake study. 

Q7. What is the importance of oral exposure?

The importance of oral exposure questions the application of current metal bioavailability models, which implicitly consider that the dominant route of exposure is via the soil solution, for bioavailability assessment and modelling of metal-based NPs. 

Q8. How many earthworms were removed from the sample?

At 0 (i.e., worms taken from batch at the start of exposure), 4, 8, 24, 36, 48, 72, 96, 120, and 168h after initiation, 4 sealed and 4 unsealed individuals were removed from containers for each of the five treatments. 

Q9. What was the effect of inhibition of the ionic treatment on the uptake of Ag?

Even though there was assimilation of Ag from soil in sealed earthworms, inhibition ofingestion resulted in a substantial reduction in uptake of both Ag forms. 

Q10. What is the effect of the oral ionic treatment on the Ag uptake?

The Ag uptake seen for the Ag NPs, both with and without the potential for oral exposure, is not in itself indicative of direct NP uptake, because assimilation may be of the ions produced by dissolution. 

Q11. What is the effect of Ag NPs on the gut?

When this organic matter is consumed, the presence of surfactant molecules and other conditions within the gut lumen may possibly allow the greater release of Ag into solution that may then be assimilated.