Uptake, Tissue Distribution, and Depuration of Total Silver in Common

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Uptake, tissue distribution, and depuration of total silver in common carp (Cyprinus carpio) after aqueous exposure to silver nanoparticles Min-Hee Jang, Woo-Keun Kim, Sung-Kyu Lee, Theodore Burdick Henry, and June-Woo Park Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5022813 • Publication Date (Web): 29 Aug 2014 Downloaded from http://pubs.acs.org on August 31, 2014

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Environmental Science & Technology

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Uptake, tissue distribution, and depuration of total silver in common carp (Cyprinus carpio)

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after aqueous exposure to silver nanoparticles

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Min-Hee Jang1, Woo-Keun Kim2, Sung-Kyu Lee2,4, Theodore B. Henry3,5, June-Woo Park2,4*

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Republic of Korea

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Republic of Korea

Future Environmental Research Center, Korea Institute of Toxicology, Jinju, 660-844,

Environmental Biology Research Center, Korea Institute of Toxicology, Jinju, 660-844,

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Technology (UST), Daejoen 305-350, Republic of Korea

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The University of Tennessee, Knoxville TN 37996, USA

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*Corresponding Author: Dr. June-Woo Park, Environmental Biology Research Center, Korea

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Institute of Toxicology, Jinju, 660-844, Republic of Korea. Tel.: 82-10-6243-8041; Fax: 82-

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55-750-3729, E-mail: [email protected]

School of Life Sciences, Heriot-Watt University, Edinburgh, Scotland, UK Human and Environmental Toxicology Program, Korea University of Science and

Department of Forestry Wildlife and Fisheries, and Center for Environmental Biotechnology,

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Keywords: uptake, biodistribution, elimination, silver nanoparticles, fish

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ACS Paragon Plus Environment


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Abstract

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Increased use and disposal of silver nanoparticles (AgNPs) has led to their release from waste

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water treatment plants into surface waters and concern over potential for negative effects in

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aquatic organisms. Investigations of AgNPs toxicity in fish have considered various species,

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exposure routes, and test endpoints; however, the toxicokinetics of total silver has not been

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studied in fish exposed to aqueous AgNPs. In this study we investigated the toxicokinetics of

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total silver in common carp (Cayprinus carpio) exposed to AgNPs [0.62±0.12 (mean±SD)

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mg L-1] for 7 days followed by a 2-week depuration period. During exposure and depuration

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fish were sampled, tissues excised (gills, brain, skeletal muscle, gastrointestinal tract, liver,

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and blood) and digested in acid, and total silver concentrations were analyzed by inductively

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coupled plasma-optical emission spectrometry (ICP-OES). Total silver in tissues increased

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during the 7-d exposure and mean concentrations were 5.61 mg kg-1, liver; 3.32 mg kg-1, gill;

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2.93 mg kg-1, gastrointestinal tract; 0.48 mg kg-1, skeletal muscle; 0.14 mg kg-1, brain; and

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0.02 mg kg-1, blood. Transmission electron microscopy (TEM) energy-dispersive

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spectroscopy (EDS) confirmed the presence of silver in the tissues. After 14 days of

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depuration, total silver returned to control levels in all tissues except liver (4.22 mg kg-1),

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gastrointestinal tract (1.26 mg kg-1), and gills (0.77 mg kg-1).

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Introduction

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Silver nanoparticles (AgNPs) are one of the most widely used metal nanoparticles

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because of their strong anti-bacterial, catalytic, and electrical properties1. These features

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make them an attractive component for many consumer products including clothing,

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cosmetics, medical appliances and other products with application of appropriate coating

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agents.1, 2 Such an increasingly widespread application of AgNPs has led to their release into

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aquatic environments, and these releases are likely to increase in the future leading to

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unknown hazards in aquatic organisms.3, 4 The toxicity of AgNPs in fish has been intensively

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studied and results indicate that toxicity might be caused by 1) silver ions released from

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AgNPs5, 6 followed by an impairment of sodium-potassium active transport7, 2) direct AgNPs

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association with biological components8 or 3) oxidative stress compounds generated by the

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AgNPs in aqueous phase.9 Absorption of AgNPs across epithelial membranes appears to be

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minimal in fish; however, AgNPs may associate with external tissue surfaces and facilitate

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release of silver ions that accumulate within internal tissues and cause toxic effects. Despite

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the numerous studies that have been conducted on AgNP toxicity in fish, there is a critical

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gap in information on Ag toxicokinetics including bioavailability, tissue distribution,

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accumulation, and depuration of total silver in fish after AgNP exposure. This information is

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essential for improving understanding of the outcome of AgNP exposure and for directing

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future toxicology studies regarding target tissues for further investigation.

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Toxicokinetics is the study of the time-course of uptake, distribution, metabolism, and

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excretion of toxicants in organisms10. Establishing toxicokinetics of total silver in the context

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of AgNP exposure is important for identification of the tissues where accumulation occurs,

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model accumulation related to exposure, and when integrated with information on

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toxicological responses, to improve AgNP risk assessments. There are a few studies that have 3



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addressed the biodistribution of AgNPs in rodent models. Kim et al. investigated the tissue

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distribution of total silver after oral exposure of AgNPs in Sprague-Dawley rats, and found a

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dose dependent accumulation in the liver, kidney and stomach.11 Lankveld et al. assessed the

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tissue distribution of total silver after intravenous administration of AgNPs of three different

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sizes and reported that the liver was a predominant site for accumulation of 80-100 nm

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AgNPs.12 These results demonstrate that the AgNPs administered to rodents can accumulate

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total silver (in forms of silver ions or nanoparticles) in the liver, kidney, and other organs.

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However, these studies could not distinguish whether the silver present in tissues was in

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particulate or ionic form because quantification was conducted by inductively coupled

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plasma mass spectrometry (ICP-MS) after complete acid digestion or tissues and ionization

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of Ag. In fish, the uptake of AgNPs has been demonstrated in early life stages such as

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embryos,13, 14 and juvenile,8 or fish-originated cells15. However, there is no report on uptake

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and elimination kinetics of total silver in adult fish exposed to AgNPs, and this information is

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required for a better understanding of the effects of these NPs in fishes. Thus, the motivation

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of the present study was to evaluate the uptake-depuration dynamics (toxicokinetics) of total

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silver among different tissues in common carp (Cyprinus carpio) after exposure to aqueous

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AgNPs. To our knowledge, this is the first study to investigate the time-dependent uptake,

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biodistribution, and elimination of total silver in fish after aqueous AgNPs exposure, and the

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results will provide quantitative information to help in the formulation of ecological and

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human health risk assessments.

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Materials and Methods

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Silver nanoparticles and characterization.

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Silver nanoparticles with an average particle diameter of 11.3 nm (citrate coated AgNPs,

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20.4% (wt/wt) aqueous solution) were purchased from ABC Nanotech Co. in Korea. The

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AgNPs contained 0.55% of the capping agent that provided stable suspension in water. The

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concentration of silver ions in the stock suspension (1,184.8 mg L-1), which was evaluated

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through ultrafiltration (Amicon centrifugal ultrafilter devices, 3kD, Millipore, Germany), was

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0.37 mg L-1 (below 0.03%, c/c). The physicochemical properties reported by manufacutrer

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were summarized in supplementary data, Table S1. Particle size and morphology were

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examined by transmission electron microscopy (TEM, TecnaiTM-G2-20 S-Twin, FEI

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Company, Hillsboro, OR, USA). Hydrodynamic diameter and zeta potential of the particles in

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the water suspensions were measured using a ZetaSizer Nano (Nano ZS 90, Malvern

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Instruments, UK). The stock suspensions were stored at 4°C in darkness until applied to the

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exposure tanks.

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Test animal and acclimation.

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Common carp (Cayprinus carpio) were purchased from a local aquaculture farm (Ochang

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fish farm, Ochang, Korea), and prior to exposure were held in 200 L tanks for >1 month

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supplied with continuously aerated tap water (23-25℃) under a 16-h light: 8-h dark

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photoperiod. Fish were fed on a maintenance ration of food (Sticksmeal, Tabia, Suwon,

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Korea) at a rate of 1% body weight per day. Fish were deprived of food for 1 day prior to the

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experiment.

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Exposure regime 5



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Twelve carps (body length of 12.7 ± 2.4 cm and weight of 28.1 ± 12.6 g) were exposed to

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AgNPs in the aqueous phase by adding AgNPs to the water column under static renewal

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conditions. Glass experimental tanks (20L) were set up in triplicate, and treatment tanks were

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dosed with 10 mL AgNPs stock suspension (1,000 mg L-1) to give nominal concentration of

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0.5 mg L-1. During the exposure, very weak aeration was supplied to the tanks to prevent the

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propensity of aggregation in the aqueous phase. In order to confirm aqueous stability of

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AgNPs, water samples were taken from each tank from the middle of the aqueous phase

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during the exposure period and total silver concentration was analyzed by inductively

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coupled plasma-optical emission spectrometry (ICP-OES, Optima 7000DV, PerkinElmer,

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USA). Fish (n = 6) were stocked into each tank and exposed to the nanoparticles for 7 days.

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Water changes of 50% were carried out every day and the tanks filled and re-dosed

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accordingly. Fish were deprived of food for the duration of exposure to avoid adsorption of

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AgNPs on the food. At 0, 1, 2, 3, 5, and 7 days fish were euthanized with ice water according

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to the Euthanasia guidelines.16 Immediately after euthanasia, the gill, gastrointestinal tract (G-

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I tract), liver, brain, fillet, and blood were excised, weighed, and stored at -80ºC until further

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analysis for quantification of accumulated total silver. During the experiment, dissolved

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oxygen, pH and temperature were monitored daily in all tanks.

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Depuration regime

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The depuration component of the study was carried out with same design of exposure regime,

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except for the addition of AgNPs. Fish (n = 6) exposed to 0.5 mg L-1 AgNPs for 7 days were

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transferred to the tanks filled with aerated clean water. Water changes of 50% were carried

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out every day to avoid re-exposure of silver excreted from fish. At 0, 1, 3, 5, 10, and 14 day

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after transfer, carp were euthanized with ice water. The gill, G-I tract, liver, brain, fillet and 6


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blood were excised, weighed, and stored at -80ºC until further analysis. During the test,

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dissolved oxygen, pH and temperature were monitored daily in all tanks.

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Total silver analyses in water and tissue samples

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The analytical procedures were validated by conducting an AgNPs spike recovery test in each

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tissue (0.5µg of AgNPs/200 mg tissue) and computation of the percent recovery of total silver

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from the tissue extracts (Table 1). Briefly, each tissue sample was placed in a 50-mL

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digestion vessel with 8 mL concentrated nitric acid (70%, Junsei, Japan) and 2 mL hydrogen

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peroxide (30%, Junsei, Japan). After complete digestion (heating at 120℃ for 4 hrs), acid

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solution was allowed to be evaporated to dryness. Then, the solid phase extract was eluted

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with ultrapure water (10 mL).

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To determine nanoparticle exposure levels in the tanks, the concentrations of AgNPs

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were measured before and after water change. Water samples (3 mL, triplicate) were taken

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from the middle of the aqueous phase and digested with 3 mL of nitric acid (30%). For the

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analysis of silver concentration in dissected tissues, approximately 200 mg of each tissue was

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applied with an open vessel acid digestion and ICP-OES quantitative analysis as described

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above. For the ICP-OES analysis, final concentration of acid in every sample was below

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10 %.

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TEM-EDS analysis

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To confirm the presence of silver in tissues, gill, liver, and G-I tract samples were fixed by

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placement on 2.5% glutaraldehyde diluted with 0.1M cacodylate buffer for 2 hr. The samples

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were then washed twice in 1x PBS buffer (pH7.4 ± 0.1) for 10 min and fixed for 1 hr in a 1:1

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mixture of 1% OsO4 and 1x PBS buffer. The samples were then rinsed with 1x PBS buffer 7



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and dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, and 99.9%). Resin

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infiltration was carried out in a graded resin series (30%, 50%, and 70%) for 2 hr each, and at

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100% overnight. After infiltration tissue, samples were embedded in beam capsules and resin

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polymerized for 72 hr at 60ºC. Semi-thin sections (150 nm) and ultra-thin sections (below 70

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nm) were prepared and imaged via TEM (TechaiTM G2 spirit Bio TWTN; FEI Company,

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Hillsboro, OR, USA). For the elemental analysis, scanning transmission electron microscopy

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(STEM), and energy dispersive X-ray spectroscopy for elemental mapping (STEM-EDS)

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were performed using a JEOL JEM model 2100F microscope (200 kV, JEM 2100F; JEOL,

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Tokyo, Japan). STEM and STEM-EDS were carried out at a camera length of 40 cm and a

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spot size of 2.4 nm. Silver and Osmium were analyzed as target elements.

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Toxicokinetic analysis

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Toxicokinetic analysis was performed on the basis of total silver concentration in each tissue

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after AgNPs exposure using the pseudo first order model, which is the classical toxicokinetics

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model.17 Elimination rate constant (

) was determined by fitting a nonlinear regression to

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the equation

is the concentration of total silver in the tissue at the

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beginning of the experiment,

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uptake rate constant (

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, where

is the concentration at time , and

is time (hrs). The

) was calculated by fitting a nonlinear regression to the equation using the values of

determined above. The half-lives (

) of

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total silver in tissues were estimated according to the equation

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blood, the concentration of total silver in tissues was extremely low and therefore

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toxicokinetic indices were not evaluated.

. In case of

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Statistical analysis

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Prior to analysis, all data sets were tested for normality (Kolmogorov-Smirnov test) and for 8


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homogeneity of variances (Levene test). Only when data were acceptable, statistical analysis

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was performed by one-way analysis of variance (ANOVA) with Tukey test to evaluate

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significant differences between tissues. The relationship between time and internal total silver

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concentrations was analyzed with Spearman’s correlation. The IBM SPSS statistics program

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(v 19.0) was used for all statistical analyses and values of p < 0.05 were considered

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significant.

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Results and Discussion

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AgNPs exposing condition

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The concentration of total silver during the exposure tests was 0.61± 0.05 mg L-1 (mean ± SD,

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n=8), which was within 20% of the nominal concentration throughout the test (Figure 1). In

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most model kinetics studies, it has been required to determine that the concentration of test

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chemical in water remains constant over the exposure period because it is able to simplify the

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estimation of relationship between aqueous concentration and organism accumulation. In our

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study design, this requirement, maintenance of aqueous concentrations of test substance, was

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satisfied.

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Total silver uptake in carp

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Total silver concentration analyses of gill, G-I tract, liver, brain, fillet, and blood indicated

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that silver accumulated in a time dependent manner without reaching to steady state (Figure

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2). Percent recoveries from tissue extracts ranged from 91.1 to 104.2 %, demonstrating that

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the open vessel acid digestion and ICP-OES analysis were appropriate to quantify the

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concentration of total silver in fish tissues (Table 1). Total silver in all tissues collected at 0

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day was found below detection limit by ICP-OES applied in the study. Over 7 days of

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exposure, the order of total silver concentration was liver > gill > G-I tract >> fillet > brain >

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blood. Fillet, brain, and blood showed very low potential to accumulate total silver compared

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to other tissues (ANOVA-post hoc tukey test, p

Uptake, Tissue Distribution, and Depuration of Total Silver in Common

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