TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via

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TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via Different Routes is Calcium-Dependent Ling-Yan Tan, Bin Huang, Shen Xu, Zhongbo Wei, Liuyan Yang, and Ai-Jun Miao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01645 • Publication Date (Web): 30 Jun 2016 Downloaded from http://pubs.acs.org on July 5, 2016

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Environmental Science & Technology

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Table of Contents Art Well-dispersed PAA-TiO2-NPs

Passive drinking

PAA-TiO2-NP aggregates

Active ingestion

Low Ca

High Ca

2 3 4

1

ACS Paragon Plus Environment


5 6 7 8 9

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TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via

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Different Routes is Calcium-Dependent

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Ling-Yan Tan, Bin Huang, Shen Xu, Zhong-Bo Wei, Liu-Yan Yang, Ai-Jun Miao*

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State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,

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Nanjing University, Nanjing, Jiangsu Province, 210023, China

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*Corresponding author: [email protected] (Email), +86 25 89680255 (Tel.), +86 25

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89680569 (Fax)

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ABSTRACT: Calcium plays versatile roles in aquatic ecosystems. In this study, we

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investigated its effects on the uptake of polyacrylate-coated TiO2 nanoparticles

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(PAA-TiO2-NPs) by the water flea (cladoceran) Daphnia magna. Particle distribution in these

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daphnids was also visualized using synchrotron radiation-based micro X-ray fluorescence

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spectroscopy, transmission electron microscopy, and scanning electron microscopy. At low

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ambient Ca concentrations in the experimental medium ([Ca]dis), PAA-TiO2-NPs were well

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dispersed and distributed throughout the daphnid; the particle concentration was highest in

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the abdominal zone and the gut, as a result of endocytosis and passive drinking of the

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nanoparticles, respectively. Further, Ca induced PAA-TiO2-NP uptake as a result of the

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increased Ca influx. At a high [Ca]dis, the PAA-TiO2-NPs formed micron-sized aggregates

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that were ingested by D. magna and concentrated only in its gut, independent of the Ca influx.

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Our results demonstrated the multiple effects of Ca on nanoparticle bioaccumulation.

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Specifically, well-dispersed nanoparticles were taken up by D. magna through endocytosis

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and passive drinking whereas the uptake of micron-sized aggregates relied on active

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ingestion.

43 44 45

3



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INTRODUCTION

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Engineered nanoparticles are particles < 100 nm in at least two dimensions.1 They are

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widely used in areas such as medicine, electronics, textiles, and environmental remediation.

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With rapid advances in nanotechnology and the increased use of nanoparticles, a substantial

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proportion will inevitably find their way into the aquatic environment. Understanding how

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nanoparticles accumulate in aquatic organisms is essential to evaluating their ecotoxicity.

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Although several studies have examined the toxicity of nanoparticles for various aquatic

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organisms,2-4 relatively little is known about their accumulation kinetics.5,6

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Water fleas are typical invertebrates universally distributed in fresh waters, and they

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play important roles in the aquatic food chain. As a representative species of water flea,

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Daphnia magna has served as a model organism in bioaccumulation studies of conventional

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and emerging pollutants.7-9 It can accumulate pollutants through dissolved uptake or dietary

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assimilation. The former applies to molecular pollutants with a sub-nano size; the main route

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of entry of these agents is the epipodites of the thoracic limbs.10 In this case, uptake is

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facilitated by a specialized cell membrane located in the gill epithelium of the daphnid that is

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presumed to participate in the active transport of molecular pollutants.11 By contrast,

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particulate pollutants are ingested by D. magna and then assimilated in its gut lumen.

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However, the route of accumulation of nanoparticles, with a size intermediate between

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molecules and bulk material, is unclear. According to current studies on nanoparticle

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bioaccumulation, it is well-established that, regardless of their physicochemical

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characteristics, a substantial proportion of nanoparticles can be accumulated in the gut of D.

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magna.12-16 Yet, whether the nanoparticles pass through the gut’s epithelial barrier is debated.

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Nanoparticles have been detected in areas outside the flea gut (e.g., in ovaries and in lipid

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storage droplets),14-16 but direct evidence that they originated from the gut is lacking. The

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possibility that nanoparticles may enter the body of Daphnia via routes other than the gut

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cannot be excluded.

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Moreover, the aggregation of nanoparticles in the aquatic environment can alter their

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bioaccumulation.17 Environmental factors such as the pH and ionic strength of the medium as

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well as the presence of cations and dissolved organic matter can strikingly influence the 4


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stability of nanoparticles.18,19 In aquatic ecosystems, for example, Ca concentrations, ranged

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from < 0.5 mg/L to > 200 mg/L, can strongly influence nanoparticle aggregation.20,21 Calcium

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is also a vital cation for D. magna, accounting for 2-8% of its dry weight, and low Ca

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concentrations adversely affect its survival and reproduction.22 At the molecular level, Ca is

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essential for many cellular processes, including endocytosis,23 which is the major cellular

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pathway of nanoparticle internalization.

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Therefore, in the present study, we examined the Ca-dependent accumulation of

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polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) in D. magna. Specifically, particle

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distribution in these daphnids at different Ca levels was visualized using synchrotron

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radiation-based micro X-ray fluorescence spectroscopy (µXRF), scanning electron

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microscopy (SEM), and transmission electron microscopy (TEM). The results showed that at

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low Ca concentrations in the experimental medium ([Ca]dis) the PAA-TiO2-NPs were well

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dispersed but at high [Ca]dis the particles readily aggregated. We then manipulated both the

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influx and the intrinsic content of Ca in daphnids and examined the impact on PAA-TiO2-NP

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uptake by D. magna. The overall objective was to elucidate the mechanisms underlying the

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effects of Ca on the bioaccumulation of PAA-TiO2-NPs.

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MATERIALS AND METHODS

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Organisms and PAA-TiO2-NPs. The cladoceran Daphnia magna and its green algal

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foods (Chlamydomonas reinhardtii and Scenedesmus obliquus) were obtained from the

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Institute of Hydrobiology, Chinese Academy of Science. The daphnids were raised in aerated

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tap water at 23.5 oC on a 12:12 h light-dark cycle with an irradiance of 30 µmol photons/m2/s.

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Their density was kept at one individual per 10 mL water, and the medium was refreshed

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every other day. Algal mixtures (2.5×104 cells/mL C. reinhardtii and 6×104 cells/mL S.

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obliquus) were fed daily to the daphnids. The diet was doubled when D. magna was older

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than 3 days. Both species of green algae were cultured in WC medium24 under the same

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environmental conditions as D. magna. In all of the experiments described below, simplified

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Elendt M7 medium (SM7)25 was the basal exposure medium and 7-day-old D. magna was the

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objective organism. 5



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The PAA-TiO2-NPs (anatase, primary particle size 1-10 nm with an isoelectric point of

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2.0) were the same as those used by Yang et al.26 and were purchased from Vive Nano

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(Toronto, Canada). The nanoparticles were coated with sodium polyacrylate (74% of total

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weight) to improve their stability in aqueous solutions. Their hydrodynamic size in the

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experimental media was determined using a dynamic light scattering particle sizer (DLS,

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ZetaPALS, Brookhaven Instruments, NY, USA).

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Forty-Eight-Hour Uptake Experiment. PAA-TiO2-NP (4.0 mg-Ti/L) uptake by D.

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magna was investigated in medium containing 0.2 and 2.0 mM Ca (in the form of CaCl2).

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The concentration of PAA-TiO2-NPs used in the experiment was far below the non-observed

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effect level (> 400 mg-Ti/L) determined in a preliminary acute toxicity study. Each treatment

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had three replicates and each replicate comprised 70 individuals. These daphnids were first

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allowed to evacuate their guts in fresh SM7 alone for 1 h and then transferred to the uptake

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medium. After 1, 2, 4, 6, 12, 24, and 48 h, 10 individuals per time point were removed and

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PAA-TiO2-NPs loosely adsorbed onto their carapaces were washed away using 3×100 mL of

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SM7. The daphnids collected at each time point were digested in 20 mL H2SO4 and 8 g

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(NH4)2SO4.27 The bioaccumulated Ti ([Ti]daphnia) was quantified using a graphite furnace

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atomic absorption spectrometer (GFAAS, Thermo Fisher Scientific Inc., Waltham, MA, USA)

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with a detection limit of 3 µg/L. The sample digestion procedure and the Ti determination

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method were verified through D. magna samples spiked with known amount of

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PAA-TiO2-NPs and a recovery of 100 ± 10% was observed.

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In another 48-h experiment, all procedures were the same except that the uptake media

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were refreshed every 12 h. The aim of this experiment was to examine whether the potential

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sedimentation of PAA-TiO2-NPs in the uptake media (especially in the treatment with 2.0 M

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Ca) influenced their accumulation during the 48-h period. Throughout both experiments, the

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concentration of PAA-TiO2-NPs suspended in the uptake media was monitored.

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Six-Hour Uptake Experiment. The procedure for this experiment was similar to that

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used in the 48-h experiment described above. However, the exposure duration was shortened

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to 6 h; three time points (1, 3, and 6 h) and five levels of [Ca]dis (0, 0.2, 0.5, 1, and 2 mM)

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were evaluated. The uptake rate of PAA-TiO2-NPs was calculated as the slope of the linear 6


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regression between [Ti]daphnia and the exposure time.26 Additionally, PAA-TiO2-NP uptake in

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the presence of 2.0 mM EGTA (ethylene glycol tetraacetic acid, a Ca-binding ligand) and at

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different pH values (6, 7, 8, and 9) was examined at various levels of [Ca]dis (0, 0.5, and 2.0

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mM in the EGTA experiment; 0, 0.2, and 2.0 mM for different pH values). As EGTA and pH

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influence Ca speciation and its influx, they may also change the uptake of PAA-TiO2-NPs by

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D. magna. In this experiment, 0.2 mM (0.5 mM) Ca was applied to maximize the potentially

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inductive (inhibitory) effect of pH (EGTA). To further explore the mechanisms underlying

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Ca-dependent PAA-TiO2-NP accumulation, the daphnids were pre-exposed to nifedipine (1

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mM), amiloride (3.0 mM), or BAY K8644 (1 and 10 µM) for 1 h before the 6-h uptake of the

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particles was examined. Nifedipine and amiloride are inhibitors of the L-type Ca channel and

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the Na/Ca (NCX) exchanger, respectively,28 whereas BAY K8644 is an agonist of

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voltage-gated Ca channels.29

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Besides the changes in ambient Ca, its speciation, and its influx, as described above, the

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intrinsic Ca content of the daphnids ([Ca]daphnia) was altered by rearing them in SM7

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containing 12.5 µM, 0.5 mM, or 5.0 mM Ca for 24 and 48 h. PAA-TiO2-NP uptake was then

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determined in SM7 at the same level of [Ca]dis (12.5 µM). The Ca content in the daphnids

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was measured by GFAAS. [Ca]daphnia, or its bioavailable concentration in the cells was further

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modulated

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[1,2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester)]. A-23187

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is a carrier of Ca; thapsigargin induces the liberation of Ca from the endoplasmic reticulum to

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the cytoplasm;30,31 and BAPTA-AM, as a strong Ca-binding ligand, enters cells by passive

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diffusion and decreases the cytosolic ionized Ca concentration.32 PAA-TiO2-NP uptake in the

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presence of 0, 0.01, 0.04, and 0.2 µM A-23187 was performed with a [Ca]dis of 0.2 mM. In

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addition, the daphnids were pre-exposed to thapsigargin (0, 0.3, 1.0, and 3.0 µM) or

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BAPTA-AM (0, 0.03, 0.2, and 1.0 mM) for 1 h, after which PAA-TiO2-NP accumulation was

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quantified in the uptake media alone, without added Ca, thapsigargin, or BAPTA-AM.

using

calcium

ionophore

(A-23187),

thapsigargin,

and

BAPTA-AM

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PAA-TiO2-NP Elimination Experiment. Seven-day-old daphnids were exposed to 4

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mg-Ti/L PAA-TiO2-NPs at a [Ca]dis of 0.2 mM for 1 h and then evenly divided into three

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depuration treatments, containing no PAA-TiO2-NPs. Daphnids in the first two treatments 7



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were transferred to fresh SM7 with and without the addition of 0.2 mM EGTA, respectively.

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Those in the third treatment were heat killed (45 oC for 2 min) and then transferred to fresh

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SM7. Each treatment consisted of three replicates, and 10 daphnids were collected from each

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one for the measurement of [Ti]daphnia after 0, 1, 3, 6, 12, 24, and 48 h of depuration.

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µXRF Studies. D. magna samples for µXRF were prepared following a procedure

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similar to that described in Laforsch and Tollrian.33 Briefly, the daphnids were exposed to 40

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mg-Ti/L PAA-TiO2-NPs for 24 h with a [Ca]dis of 0.2 and 2.0 mM. They were then fixed in

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4% methanol for 10 min before being dehydrated in graded acetone solutions (70, 80, 90,

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2×98, and 2×100%) for 10 min each. Subsequently, the specimens were immersed in 1.5 mL

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of HMDS (1,1,1,3,3,3 hexamethyldisilazane); 90% of the HMDS was pipetted out after 30

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min, and the rest was evaporated in a desiccator. The PAA-TiO2-NP concentrations used in

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this and the experiments described below were ten times higher than the concentrations used

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in the uptake and elimination experiments, because of the detection limit of µXRF, TEM, and

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SEM. Nevertheless, the average hydrodynamic size of PAA-TiO2-NPs remained unchanged.

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µXRF mapping of Ti (KL3, 4.5109 keV) and Ca (KL3, 3.6917 keV) in the daphnids was

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performed using the BL15U beamline at the Shanghai Radiation Synchrotron Facility

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(Shanghai, China). The storage ring current was 200-300 mA with an energy level of 3.5

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GeV. Element maps were obtained by scanning the samples with a 10-keV monochromatic

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beam, which was focused to 50 × 50 µm2 using K-B optics. The step size and scanning time

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were 50 µm and 3 s, respectively. X-ray fluorescence was recorded using the 7-element Si (Li)

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detector combined with a multiple channel analyzer (e2v, UK). The fluorescence data were

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processed using Pviewer (version 1.0) and 2D Array Image Data Plotter (version 1.0).

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TEM and SEM Analyses. A procedure similar to that described in a previous study by

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our group27 was used to process the daphnids. Briefly, after a 24-h exposure to 40 mg-Ti/L

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PAA-TiO2-NPs, the fleas were fixed in 3% glutaraldehyde prepared in 0.2 M

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phosphate-buffered saline (PBS). They were then cleaned with PBS, stained in 1% osmium

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tetroxide, and dehydrated sequentially in acetone solutions (30, 50, 70, 80, 90, and 2×100%).

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Afterward, they were embedded into epoxy resin, sectioned to 100-nm thickness, and stained

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with uranyl acetate and lead citrate. The elemental composition of the potentially 8


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PAA-TiO2-NP-containing spots in the TEM samples was determined using an

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energy-dispersive X-ray (EDX) spectrometer (JEM-2100, JEOL, Tokyo, Japan). For SEM

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analysis, the D. magna samples were fixed and dehydrated following the same procedure as

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that for µXRF. After the carapace was removed using Teflon forceps, the thoracic limbs were

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collected and their Ti distribution was mapped by SEM-EDX (S-4800, Hitachi, Tokyo,

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Japan).

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Statistical Analysis. Significant differences were defined based on a p value of < 0.05,

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obtained in a one-way or two-way analysis of variance with posthoc multiple comparisons

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(Tukey or Tamhane; SPSS 11.0 by SPSS, Chicago, USA). The analysis of variance took into

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account both the normality (Kolmogorov-Smirnov and Shapiro-Wilk tests) and the

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homogeneity of variance (Levene’s test) of the data.

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RESULTS AND DISCUSSION

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Ca Effects on PAA-TiO2-NP Accumulation. According to the conventional

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biodynamic model,34 the content of a pollutant in an organism (Cb) first increases with

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exposure time (t) and then levels off, as described by Eq. (1):

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Cb =

k u C w (1 − e − ( ke + µ )t ) ke + µ

(1)

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where Cw represents the pollutant’s concentration in the uptake medium, ku and ke are the

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uptake and efflux rate constants, respectively, and µ is the organism’s growth rate, which is

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negligible for D. magna.34 In the present study, a hyperbolic relationship between Cb and t

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was not observed when the medium in the 48-h uptake experiment was not regularly

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refreshed (Figure 1a). Thus, [Ti]daphnia increased initially with exposure time, reached its

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maximum after 12 h, and decreased thereafter at both [Ca]dis (0.2 and 2.0 mM). According to

212

Eq. (1), a hyperbolic relationship between Cb and t requires that ku, ke, and Cw remain

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constant during the experimental period. When the uptake medium was refreshed every 12 h,

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[Ti]daphnia in the high-Ca treatment leveled off instead of decreasing after reaching a

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maximum (Figure 1a). This finding implies that the decrease in Ti accumulation after a 12-h

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exposure at high Ca levels was primarily due to the reduced concentration of PAA-TiO2-NPs 9



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suspended in the uptake medium. This explanation is supported by the formation of

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PAA-TiO2-NP aggregates with an average size of 1650 nm, which after 12 h settled on the

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bottom of the container, thus reducing the concentration (4 mg-Ti/L) of suspended

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PAA-TiO2-NPs by > 70%. Similarly, quantum dot concentration in the gut of D. magna was

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also found to decrease as a result of the decrease in the water column quantum dot

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concentration.35 Unlike in the high-Ca (2.0 mM) treatment, the pattern of [Ti]daphnia variation

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with exposure time remained unchanged, regardless of whether the medium was refreshed, at

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a [Ca]dis of 0.2 mM. In this case, PAA-TiO2-NPs were well dispersed, with an average size of

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20 nm, and their suspended concentration remained stable even when the uptake medium was

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not refreshed. Therefore, the parabolic relationship between [Ti]daphnia and the exposure time

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in the low-Ca treatment was due to the change in ku, ke, or both. It appears that D. magna has

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a regulation system to limit excessive accumulation of PAA-TiO2-NPs, similar to what was

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described in Adam et al.36. These results also suggested that, at the two [Ca]dis, the

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PAA-TiO2-NPs were taken up by D. magna through disparate routes, which further implied

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that the nanoparticle uptake pathway might be aggregation/size dependent. This will be

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discussed below. Nevertheless, it is also likely that the PAA-TiO2-NP uptake route was the

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same, but their distribution in daphnids or their efflux was Ca-dependent.

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In addition to the different patterns of [Ti]daphnia variation with exposure time,

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Ca-related effects on nanoparticle accumulation were evidenced by the significantly (p < 0.05)

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higher [Ti]daphnia when [Ca]dis was 2.0 mM (Figure 1a). To determine whether this

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accumulation disparity was simply caused by Ca-induced aggregation, PAA-TiO2-NP uptake

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rates at five Ca levels were compared (Figure 1b). As [Ca]dis increased from 0 to 0.5 mM, the

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hydrodynamic size (19.4-20.4 nm) of the PAA-TiO2-NPs remained stable, but their uptake

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rate was significantly (p < 0.05) increased, by 243%. When [Ca]dis was further enhanced to

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1.0 and 2.0 mM, massive aggregation occurred, yielding particle sizes of 675.4 and 1561.1

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nm, respectively. The uptake rates of PAA-TiO2-NPs at these two highest Ca levels were

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comparable. It seemed that Ca induced the uptake of well-dispersed PAA-TiO2-NPs but had

244

no effect on their micron-sized aggregates. This also suggested different routes of entry for

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well-dispersed PAA-TiO2-NPs and their micron-sized aggregates in D. magna, a hypothesis 10


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later supported by the difference in the distribution of Ti in daphnids at a [Ca]dis of 0.2 vs. 2.0

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mM (Figure 2). The D. magna in the different treatments was distinguished by the Ca signal

248

(Figure 2a, c, and e), with a negligible amount of Ti in unexposed daphnids (Figure 2b). At a

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[Ca]dis of 0.2 mM, a substantial amount of Ti accumulated within the daphnids (Figure 2d),

250

mostly in the gut and abdominal zone. Separating these two sites was a region with a

251

relatively low Ti content. Thus, the PAA-TiO2-NPs concentrated in these two tissues may

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have differed in their origins. Nanoparticle accumulation in the abdominal zone was also

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evidenced by the pronounced Ti signal in the epipodite and filter setae isolated from the

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bodies of daphnids (Figure 3a, b). In contrast to the low-Ca treatment (Figure 2d), the

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PAA-TiO2-NPs were detected only in the daphnid gut when [Ca]dis was 2.0 mM (Figure 2f).

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According to the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory,37,38 nanoparticles

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aggregate rapidly at high Ca levels due to the screening of their surface charge and the lower

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aggregation energy barrier. The distance between filtering setae on Daphnia thoracic limbs is

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approximately 0.5 µm (Figure 3a), and filtration, rather than electrostatic attraction, is the

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main mechanism for particle capture by daphnids.10,39 This explains the accumulation of the

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micron-sized PAA-TiO2-NP aggregates in the gut. In contrast to their micron-sized

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counterparts, well-dispersed PAA-TiO2-NPs with a size of ~20 nm cannot be taken up by

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daphnids through setae filtration, which suggests a route other than ingestion. Gophen and

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Geller40 found that particles < 0.5 µm did not accumulate in the daphnid gut. Nevertheless,

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our detection of the low-level gut accumulation of well-dispersed PAA-TiO2-NPs at low Ca

266

levels suggests passive drinking (also called incidental ingestion) of the experimental

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medium by the flea.10 A substantial accumulation of well-dispersed gold nanoparticles in the

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gut tract of D. magna was also reported in Wray and Klaine.8 They further proposed that this

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phenomenon was caused by incidental ingestion through physicochemical processes such as,

270

gravitational deposition, inertial impaction, motile-particle deposition, and electrostatic

271

interaction.

272

That the Ti signal in the D. magna abdominal zone at a low [Ca]dis reflected true

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internalization of the well-dispersed PAA-TiO2-NPs rather than simply their attachment to the

274

thoracic limb surface was supported by the following observations. When the 11



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nanoparticle-containing daphnids were transferred to fresh SM7, a biphasic decrease in

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[Ti]daphnia was observed during the 48-h depuration period (Figure 4). After 12 h, > 70% of the

277

PAA-TiO2-NPs were depurated, and only 15% was retained at 48 h. The presence of EGTA in

278

the

279

PAA-TiO2-NP-containing daphnids were killed at the beginning of the experiment, efflux was

280

not significant (p > 0.05). Together, these findings suggested that the Ca-mediated induction

281

of the accumulation of well-dispersed PAA-TiO2-NPs was not simply due to the increased

282

adsorption of the particles onto the daphnids and that depuration was not a passive process

283

occurring by, e.g., surface desorption. Otherwise, EGTA might accelerate the depuration of

284

PAA-TiO2-NPs and a notable elimination of PAA-TiO2-NPs by the heat-killed daphnids

285

might be observed. Therefore, the notable Ti signal in Figure 2d reflected the internalization

286

of the PAA-TiO2-NPs. This conclusion was further supported by the two-orders-of-magnitude

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lower accumulation of Ti in heat-killed (0.02-0.06 mg-Ti/g-dw) than in living daphnids.

288

Moreover, TEM images of tissue slices from D. magna abdominal zone showed a substantial

289

internalization of PAA-TiO2-NPs into epipodite cells (Figure 3c, d), where they were

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primarily concentrated in micron-sized endosomes. Research in daphnids, although limited,

291

has shown that accumulated nanoparticles are either confined to the gut lumen or pass

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through the gut’s epithelial barrier to specific anatomic sites, such as the ovaries and

293

lipid-storage droplets. Heinlaan et al.12 stated that the implicit internalization of CuO

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nanoparticles via D. magna midgut epithelial cells was not obvious. Khan et al.13 found no

295

evidence for trans-epithelial alimentary uptake of ingested Au nanoparticles by D. magna

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either. By contrast, fluorescent polystyrene nanoparticles were observed to accumulate in D.

297

magna lipid-storage droplets, suggesting their penetration of gut epithelial cells.14 Similarly,

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quantum dots of various surface coatings concentrated in the D. magna gastrointestinal tract,

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brood chamber, and exoskeleton.15 Nevertheless, direct evidence for the translocation of these

300

nanoparticles from the gut to the objective location is lacking. Other entry routes may thus

301

exist, as supported not only by the results of the present study but also by those of other

302

researchers. For example, although Ag nanoparticles accumulated in D. magna ovaries,16

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there was no evidence that they had penetrated the gastrointestinal tract barrier and their

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concentration in the gut lumen was low.

efflux

medium

had

no

effect

on

depuration.

12


By

contrast,

when

the

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Manipulation of Ambient Ca Speciation and Its Influx. To further elucidate the

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mechanisms underlying the Ca-dependent accumulation of well-dispersed PAA-TiO2-NPs

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and to confirm different uptake routes of well-dispersed PAA-TiO2-NPs and their

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micron-sized aggregates, the effects of EGTA, pH, and Ca channel inhibitors/agonist on

309

PAA-TiO2-NP uptake were investigated. A pre-requisite of these experiments is that the

310

nanoparticle aggregation status was unaffected by EGTA, pH, and Ca channel

311

inhibitors/agonist. In the presence of 2.0 mM EGTA, free Ca ions in the medium were

312

reduced dramatically, such that they contributed < 1% to the total Ca. Under this condition,

313

PAA-TiO2-NP uptake was suppressed by 65.7% when the [Ca]dis was 0.5 mM, whereas

314

inhibition was not significant (p > 0.05) at a [Ca]dis of 2.0 mM (Figure 5a). PAA-TiO2-NP

315

uptake suppression in the first case was not caused by EGTA itself or by cations other than Ca

316

(e.g., K, Na, and Mg), considering the fact that neither EGTA nor EDTA, as common

317

metal-binding ligands, influenced the uptake of well-dispersed PAA-TiO2-NPs in the absence

318

of Ca. Moreover, PAA-TiO2-NPs were well-dispersed with an average particle size of 20 nm

319

and their uptake remained constant in medium containing 0-1.0 mM Mg but without Ca

320

(Supporting Information, Figure S1). All these phenomena imply that the uptake of

321

well-dispersed PAA-TiO2-NPs is Ca-dependent rather than a general effect of cations and this

322

process is determined by the concentration of free Ca ions rather than the total Ca

323

concentration.

324

The results of the EGTA experiment suggested two possible mechanisms for the

325

observed effects of free Ca ions. (1) The free Ca ion concentration determines the amount of

326

Ca adsorbed on the surfaces of PAA-TiO2-NPs and D. magna, which in turn influences

327

attachment of the nanoparticles to the target sites and their subsequent internalization.41 A

328

positive correlation between cations such as Ca, Mg, and Na and the attachment efficiency of

329

nanoparticles (e.g., fullerene and α-Fe2O3) was previously reported.21,41 (2) The influx of Ca

330

and its bioavailability in the cytosol or certain micro-domains of D. magna cells depend on its

331

ambient free ion concentration. Endocytosis, as the main form of cellular nanoparticle

332

internalization, has been related to the influx or intracellular content of Ca. Lew et al.42

333

showed that Fc-receptor-triggered phagocytosis in human neutrophils depends on 13



334

intracellular Ca transients. Our preliminary experiment in the protozoan Tetrahymena

335

thermophila also demonstrated that Ca induced the endocytosis of well-dispersed

336

PAA-TiO2-NPs (Supporting Information, Figure S2). In a study in rodents, Ca influx initiated

337

all forms of endocytosis at a single nerve terminal in response to the speeding up of

338

membrane invagination and fission.43 The Ca sensor mediating these forms of endocytosis is

339

calmodulin, which activates the phosphatase calcineurin and thus targets numerous endocytic

340

proteins.23

341

To determine which of the two mechanisms proposed above is the more likely one in D.

342

magna, PAA-TiO2-NP uptake at different pH values but with the same [Ca]dis was compared.

343

At the four pH values tested (6-9), the free Ca ion concentration in the medium remained

344

constant for each [Ca]dis, particularly at pH 6, 7, and 8. Nevertheless, when [Ca]dis was 0.2

345

mM, the PAA-TiO2-NP uptake rate increased gradually, from 0.28 mg-Ti/g-dw/h at pH 6 to

346

0.86 mg-Ti/g-dw/h at pH 8 and leveled off thereafter (Figure 5b). By contrast, when [Ca]dis

347

was 0 or 2.0 mM, PAA-TiO2-NP uptake was independent of ambient pH. This discrepancy

348

implied that Ca plays an important role in the effects of pH on the uptake of well-dispersed

349

PAA-TiO2-NPs. According to the free ion activity model (FIAM) as proposed for aquatic

350

organisms,44 Ca adsorption and its uptake should increase with increasing pH, as the

351

competition from H+ ions decreases. It seems that the uptake of well-dispersed

352

PAA-TiO2-NPs was related to Ca influx. Nevertheless, Ca adsorption on PAA-TiO2-NPs, as

353

measured by GFAAS, also rose from 520 to 628 mg g-Ti-1 as the pH was increased from 6 to

354

9, although it was less influenced by pH than by EGTA (248 and 570 mg g-Ti-1 of Ca on

355

PAA-TiO2-NPs in the presence and absence of 2.0 mM EGTA). Therefore, the first possibility

356

of the above-proposed mechanisms cannot be excluded.

357

As inhibitors of the L-type Ca channel and the NCX exchanger, nifedipine and

358

amiloride inhibit Ca uptake.20 Although neither inhibitor altered Ca speciation in the medium

359

or its adsorption onto PAA-TiO2-NPs and the D. magna surface, at a [Ca]dis of 0.5 mM

360

PAA-TiO2-NP uptake decreased by 33.9 and 79.8% in the presence of nifedipine and

361

amiloride, respectively (Figure 5c). By contrast, at a [Ca]dis of 2.0 mM the effects were not

362

significant (p > 0.05), with PAA-TiO2-NP uptake remaining in the range of 0.96-1.38 14


Page 14 of 28

Page 15 of 28


363

mg-Ti/g-dw/h. Similar to the high-Ca treatment, nifedipine did not suppress PAA-TiO2-NP

364

uptake in medium without added Ca; amiloride, however, reduced PAA-TiO2-NP uptake by

365

74.5%, suggesting that its effects were Ca-independent. In fact, amiloride also inhibits

366

Na+/H+ exchangers and may therefore limit endocytosis by lowering the submembranous pH

367

and preventing Rac1 and Cdc42 signaling.45 Nevertheless, the possibility that amiloride

368

altered Ca translocation between the different compartments of D. magna cells and thereby

369

reduced the intracellularly bioavailable Ca cannot be excluded. As for the Ca channel agonist

370

BAY K8644, its effect on PAA-TiO2-NP uptake was dependent on [Ca]dis, with induction

371

observed at 0.2 mM but not at 0 or 2.0 mM (Figure 5d). Collectively, the results from the

372

inhibitor and agonist experiments clearly demonstrated that Ca influx or its bioavailable

373

concentration in D. magna cells plays a critical role in the uptake of well-dispersed

374

PAA-TiO2-NPs. Further, the completely different responses of the uptake of well-dispersed

375

PAA-TiO2-NPs and their micron-sized aggregates to EGTA, pH, nifedipine, amiloride, and

376

BAY K8644 also suggested their different routes of entry in daphnids.

377

Effects of Intracellular Ca Bioavailability. We then asked whether the Ca-dependent

378

uptake of well-dispersed PAA-TiO2-NPs was attributable to the change in Ca influx itself or

379

to fluctuations of its bioavailability in the cytosol or certain micro-domains of D. magna cells.

380

To answer this question, [Ca]daphnia was manipulated as follows. When the daphnids were

381

pre-cultured in media with a [Ca]dis of 0.0125, 0.5, and 5 mM, [Ca]daphnia was, respectively,

382

36.3, 37.2, and 49.3 mg/g after 24 h and 18.3, 37.5, and 42.8 mg/g after 48 h (Figure 6a) as

383

measured by GFAAS. The daphnids look healthy at all three Ca levels based on their

384

swimming behavior. At both time points, the absence of a significant (p > 0.05) alteration of

385

PAA-TiO2-NP uptake could be explained by the fact that [Ca]dis (0.0125 mM) was the same

386

in the uptake media of the different treatments such that Ca influx was the same for the

387

different Ca-containing daphnids. Alternatively, although [Ca]daphnia changed, how the

388

intracellular Ca speciation may vary was unclear and thus the free Ca concentration in the

389

cytosol or other micro-domains of the daphnids may have remained unchanged. In addition to

390

allowing the daphnids to acclimate to media with different [Ca]dis, the lipophilic Ca-binding

391

reagent BAPTA-AM was applied to reduce the intracellular concentration of free Ca ions. No 15



392

significant (p > 0.05) effects were found in either case (Figure 6b). When the Ca

393

concentration in the cytosol of daphnids was increased by the addition of a calcium ionophore

394

or thapsigargin, particle uptake was unaffected (Figure 6b). Taken together, these results

395

suggest that the influx of Ca through the plasma membrane, but not its concentration in

396

daphnids, determines the uptake of well-dispersed PAA-TiO2-NPs. Moreover, the uptake of

397

dissolved Ca occurred primarily in D. magna epipodites, where well-dispersed

398

PAA-TiO2-NPs concentrate,10 suggesting that the nanoparticles are taken up by the thoracic

399

limbs. During the Ca transients that occur in all organisms, local Ca concentrations may

400

increase to tens of µM upon the opening of Ca channels and then fall dramatically during

401

their closure.46 Therefore, it is also likely that in a multicellular organism such as D. magna

402

the addition of Ca ionophore, thapsigargin, or BAPTA-AM did not change the Ca

403

concentration in the specific micro-domains responsible for PAA-TiO2-NP uptake.

404

Overall, our study demonstrated the multiple effects of Ca on PAA-TiO2-NP

405

accumulation. When the Ca level in the medium was low (0-0.5 mM), the PAA-TiO2-NPs

406

were well dispersed and their hydrodynamic size was approximately 20 nm. In this case,

407

passive drinking and endocytosis were the dominant routes for PAA-TiO2-NP accumulation.

408

The nanoparticles taken up by these two routes were distributed throughout the daphnids,

409

with the highest concentrations in the abdominal zone and gut as a result of endocytosis and

410

passive drinking, respectively. When the Ca level in the medium was high (≥ 1 mM), the

411

PAA-TiO2-NPs formed micron-sized aggregates that were actively ingested by D. magna,

412

such that they were exclusively located in the gut. In addition to the effects of [Ca]dis on

413

nanoparticle aggregation and uptake route, the influx of Ca and possibly its concentration in

414

specific micro-domains of the organism could facilitate the uptake of well-dispersed

415

PAA-TiO2-NPs. The Ca-dependent uptake and distribution of PAA-TiO2-NPs we observed

416

herein are likely able to be extended to other nanoparticles and other aquatic organisms.

417

Therefore, the uptake pathways identified in the present study should be considered in risk

418

evaluations of nanoparticles.

419

420

ACKNOWLEDGEMENTS We thank three anonymous reviewers and Dr. Qiaoguo Tan for their instructive 16


Page 16 of 28

Page 17 of 28


421

comments on this paper. The financial support offered by Chinese public science and

422

technology research funds projects of ocean (grant no. 201505034) and by the National

423

Natural Science Foundation of China (grant nos. 41271486, 41001338, and 21237001) to A. J.

424

Miao made this work possible.

425

SUPPORTING INFORMATION

426

Additional figures showing PAA-TiO2-NP uptake at different levels of Mg as well as the

427

effects of EGTA on PAA-TiO2-NP uptake by Tetrahymena thermophila are included. This

428

material is available free of charge on the ACS Publications Website.

429 430

17



431

432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473

(1) Klaine, S. J.; Alvarez, P. J. J.; Batley, G. E.; Fernandes, T. F.; Handy, R. D.; Lyon, D. Y.; Mahendra, S.; McLaughlin, M. J.; Lead, J. R. Nanomaterials in the environment: Behavior, fate, bioavailability, and effects. Environ. Toxicol. Chem. 2008, 27, (9), 1825-1851. (2) Bondarenko, O.; Juganson, K.; Ivask, A.; Kasemets, K.; Mortimer, M.; Kahru, A. Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: A critical review. Arch. Toxicol. 2013, 87, (7), 1181-1200. (3) Soni, D.; Naoghare, P. K.; Saravanadevi, S.; Pandey, R. A. Release, transport and toxicity of engineered nanoparticles. In Reviews of Environmental Contamination and Toxicology; Whitacre, D. M., Ed.; Springer International Publishing: New York City 2015; pp 1-47. (4) Nam, D. H.; Lee, B. C.; Eom, I. C.; Kim, P.; Yeo, M. K. Uptake and bioaccumulation of titanium- and silver-nanoparticles in aquatic ecosystems. Mol. Cell. Toxicol. 2014, 10, (1), 9-17. (5) Wang, Y.; Miao, A. J.; Luo, J.; Wei, Z. B.; Zhu, J. J.; Yang, L. Y. Bioaccumulation of CdTe quantum dots in a freshwater alga Ochromonas danica: A kinetics study. Environ. Sci. Technol. 2013, 47, (18), 10601-10610. (6) Croteau, M. N.; Misra, S. K.; Luoma, S. N.; Valsami-Jones, E. Bioaccumulation and toxicity of CuO nanoparticles by a freshwater invertebrate after waterborne and dietborne exposures. Environ. Sci. Technol. 2014, 48, (18), 10929-10937. (7) Zhao, C. M.; Wang, W. X. Biokinetic uptake and efflux of silver nanoparticles in Daphnia magna. Environ. Sci. Technol. 2010, 44, (19), 7699-7704. (8) Wray, A. T.; Klaine, S. J. Modeling the influence of physicochemical properties on gold nanoparticle uptake and elimination by Daphnia magna. Environ. Toxicol. Chem. 2015, 34, (4), 860-872. (9) Li, W. M.; Wang, W. X. Distinct biokinetic behavior of ZnO nanoparticles in Daphnia magna quantified by synthesizing Zn-65 tracer. Water Res. 2013, 47, (2), 895-902. (10) Smirnov, N. Physiology of the Cladocera. Academic Press: Cambridge, Massachusetts, USA, 2013. (11) Kikuchi, S. A unique cell-membrane with a lining of repeating subunits on the cytoplasmic side of presumably ion-transporting cells in the gill epithelium of Daphnia magna (Crustacea, Cladocera). J. Submicr. Cytol. Path. 1982, 14, (4), 711-715. (12) Heinlaan, M.; Kahru, A.; Kasemets, K.; Arbeille, B.; Prensier, G.; Dubourguier, H. C. Changes in the Daphnia magna midgut upon ingestion of copper oxide nanoparticles: A transmission electron microscopy study. Water Res. 2011, 45, (1), 179-190. (13) Khan, F. R.; Kennaway, G. M.; Croteau, M. N.; Dybowska, A.; Smith, B. D.; Nogueira, A. J. A.; Rainbow, P. S.; Luoma, S. N.; Valsami-Jones, E. In vivo retention of ingested Au NPs by Daphnia magna: No evidence for trans-epithelial alimentary uptake. Chemosphere 2014, 100, 97-104. (14) Rosenkranz, P.; Chaudhry, Q.; Stone, V.; Fernandes, T. F. A comparison of nanoparticle and fine particle uptake by Daphnia magna. Environ. Toxicol. Chem. 2009, 28, (10), 2142-2149. (15) Feswick, A.; Griffitt, R. J.; Siebein, K.; Barber, D. S. Uptake, retention and

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internalization of quantum dots in Daphnia is influenced by particle surface functionalization. Aquat. Toxicol. 2013, 130, 210-218. (16) Georgantzopoulou, A.; Balachandran, Y. L.; Rosenkranz, P.; Dusinska, M.; Lankoff, A.; Wojewodzka, M.; Kruszewski, M.; Guignard, C.; Audinot, J. N.; Girija, S.; Hoffmann, L.; Gutleb, A. C. Ag nanoparticles: size- and surface-dependent effects on model aquatic organisms and uptake evaluation with NanoSIMS. Nanotoxicology 2013, 7, (7), 1168-1178. (17) Kwon, D.; Jeon, S. K.; Yoon, T. H. Impact of agglomeration on the bioaccumulation of sub-100 nm sized TiO2. Colloid. Surface. B 2014, 116, 277-283. (18) Navarro, E.; Baun, A.; Behra, R.; Hartmann, N. B.; Filser, J.; Miao, A. J.; Quigg, A.; Santschi, P. H.; Sigg, L. Environmental behavior and ecotoxicity of engineered nanoparticles to algae, plants, and fungi. Ecotoxicology 2008, 17, (5), 372-386. (19) Quigg, A.; Chin, W. C.; Chen, C. S.; Zhang, S.; Jiang, Y.; Miao, A. J.; Schwehr, K. A.; Xu, C.; Santschi, P. H. Direct and indirect toxic effects of engineered nanoparticles on algae: Role of natural organic matter. ACS Sustainable Chem. Eng. 2013, 1, (7), 686-702. (20) Tan, Q. G.; Wang, W. X. Interspecies differences in calcium content and requirement in four freshwater cladocerans explained by biokinetic parameters. Limnol. Oceanogr. 2010, 55, (3), 1426-1434. (21) Chen, K. L.; Mylon, S. E.; Elimelech, M. Aggregation kinetics of alginate-coated hematite nanoparticles in monovalent and divalent electrolytes. Environ. Sci. Technol. 2006, 40, (5), 1516-1523. (22) Tan, Q. G.; Wang, W. X. The regulation of calcium in Daphnia magna reared in different calcium environments. Limnol. Oceanogr. 2009, 54, (3), 746-756. (23) Wu, L. G.; Hamid, E.; Shin, W.; Chiang, H. C. Exocytosis and endocytosis: Modes, functions, and coupling mechanisms. Ann. Rev. Physiol. 2014, 76, 301-331. (24) Guillard, R. R.; Lorenzen, C. J. Yellow-green algae with chlorophyllide C. J. Phycol. 1972, 8, (1), 10-14. (25) Miao, A. J.; Wang, N. X.; Yang, L. Y.; Wang, W. X. Accumulation kinetics of arsenic in Daphnia magna under different phosphorus and food density regimes. Environ. Toxicol. Chem. 2012, 31, (6), 1283-1291. (26) Yang, W. W.; Wang, Y.; Huang, B.; Wang, N. X.; Wei, Z. B.; Luo, J.; Miao, A. J.; Yang, L. Y. TiO2 nanoparticles act as a carrier of Cd bioaccumulation in the ciliate Tetrahymena thermophila. Environ. Sci. Technol. 2014, 48, (13), 7568-7575. (27) Yang, W. W.; Miao, A. J.; Yang, L. Y. Cd2+ toxicity to a green alga Chlamydomonas reinhardtii as influenced by its adsorption on TiO2 engineered nanoparticles. Plos One 2012, 7, (3) e32300. (28) Granado e Sa, M.; Baptista, B. B.; Farah, L. S.; Leite, V. P.; Zanotto, F. P. Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei. J. Comp. Physiol. B 2010, 180, (3), 313-321. (29) Benaim, G.; Garcia-Marchan, Y.; Reyes, C.; Uzcanga, G.; Figarella, K. Identification of a sphingosine-sensitive Ca2+ channel in the plasma membrane of Leishmania mexicana. Biochem. Bioph. Res. Co. 2013, 430, (3), 1091-1096. (30) Verma, A.; Bhatt, A. N.; Farooque, A.; Khanna, S.; Singh, S.; Dwarakanath, B. S. Calcium ionophore A23187 reveals calcium related cellular stress as "I-Bodies": An old actor in a new role. Cell Calcium 2011, 50, (6), 510-522. 19



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(31) Lytton, J.; Westlin, M.; Hanley, M. R. Thapsigargin inhibits the sarcoplasmic or endoplasmic-reticulum Ca-ATPase family of calcium pumps. J. Biol. Chem. 1991, 266, (26), 17067-17071. (32) Cousin, M. A.; Robinson, P. J. Ca2+ influx inhibits dynamin and arrests synaptic vesicle endocytosis at the active zone. J. Neurosci. 2000, 20, (3), 949-957. (33) Laforsch, C.; Tollrian, R. A new preparation technique of daphnids for Scanning Electron Microscopy using hexamethyldisilazane. Arch. Hydrobiol. 2000, 149, (4), 587-596. (34) Tsui, M. T. K.; Wang, W. X. Biokinetics and tolerance development of toxic metals in Daphnia magna. Environ. Toxicol. Chem. 2007, 26, (5), 1023-1032. (35) Jackson, B. P.; Pace, H. E.; Lanzirotti, A.; Smith, R.; Ranville, J. F. Synchrotron X-ray 2D and 3D elemental imaging of CdSe/ZnS quantum dot nanoparticles in Daphnia magna. Anal. Bioanal. Chem. 2009, 394, (3), 911-917. (36) Adam, N.; Leroux, F.; Knapen, D.; Bals, S.; Blust, R. The uptake and elimination of ZnO and CuO nanoparticles in Daphnia magna under chronic exposure scenarios. Water Res. 2015, 68, 249-261. (37) Derjaguin, B.; Landau, L. Theory of stability of highly charged liophobic sols and adhesion of highly charged particles in solutions of electrolytes. Zh. Eksp. Teor. Fiz. 1945, 15, (11), 663-682. (38) Verwey, E. J. W.; Overbeek, J. T. G. Theory of the Stability of Lyophobic Colloids; Elsevier: Amsterdam, 1948. (39) Fryer, G. Functional morphology and the adaptive radiation of the Daphniidae (Branchipoda, Anomopoda). Philos. T. Roy. Soc. B 1991, 331, (1259), 1-99. (40) Gophen, M.; Geller, W. Filter mesh size and food particle uptake by Daphnia. Oecologia 1984, 64, (3), 408-412. (41) Chen, K. L.; Elimelech, M. Aggregation and deposition kinetics of fullerene (C-60) nanoparticles. Langmuir 2006, 22, (26), 10994-11001. (42) Lew, D. P.; Andersson, T.; Hed, J.; Divirgilio, F.; Pozzan, T.; Stendahl, O. Ca2+-dependent and Ca2+-independent phagocytosis in human-neutrophils. Nature 1985, 315, (6019), 509-511. (43) Wu, X. S.; McNeil, B. D.; Xu, J.; Fan, J.; Xue, L.; Melicoff, E.; Adachi, R.; Bai, L.; Wu, L. G. Ca2+ and calmodulin initiate all forms of endocytosis during depolarization at a nerve terminal. Nat. Neurosci. 2009, 12, (8), 1003-1010. (44) Campbell, P. G. C. Interactions between trace metals and aquatic organisms: A critique of the free-ion activity model. In Metal Speciation and Bioavailability in Aquatic Systems; Tessier, A., Turner, D. R., Eds.; John Wiley & Sons Ltd.: Chichester, UK, 1995; pp 45-102. (45) Koivusalo, M.; Welch, C.; Hayashi, H.; Scott, C. C.; Kim, M.; Alexander, T.; Touret, N.; Hahn, K. M.; Grinstein, S. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J. Cell Biol. 2010, 188, (4), 547-563. (46) Hosoi, N.; Holt, M.; Sakaba, T. Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse. Neuron 2009, 63, (2), 216-229

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561

Figure Legends

562

Figure 1. (a) Variations in the Ti content in Daphnia magna ([Ti]daphnia) with exposure time at

563

ambient Ca concentrations ([Ca]dis) of 0.2 and 2.0 mM, as determined in a 48-h uptake

564

experiment. The medium was either refreshed (0.2-re and 2-re) or not (0.2 and 2). (b)

565

PAA-TiO2-NP uptake rates at different [Ca]dis during a 6-h uptake experiment. The values of

566

the bars with different letters on the top are statistically significant (p < 0.05) from each other.

567

Data are the mean ± standard deviation (n = 3).

568

Figure 2. Distribution of (a, c, e) Ca and (b, d, f) Ti in Daphnia magna (a, b) during a control

569

treatment in the absence of PAA-TiO2-NPs or (c-f) when pre-exposed to 40 mg-Ti/L

570

PAA-TiO2-NPs for 3 h with a [Ca]dis of (c, d) 0.2 and (e, f) 2.0 mM, as determined by

571

synchrotron radiation-based micro X-ray fluorescence spectroscopy (µXRF). White arrows

572

indicate the accumulation of PAA-TiO2-NPs in the gut (GT) and abdominal area (AD) of D.

573

magna.

574

Figure 3. (a) Scanning electron microcopy (SEM) image of the thoracic limb of Daphnia

575

magna pre-exposed to 40 mg-Ti/L PAA-TiO2-NPs for 24 h. (b) Ti mapping (red dots) of the

576

thoracic limb using SEM-EDX. Arrows indicate the areas with a high Ti signal. (c)

577

Distribution of PAA-TiO2-NPs in cells captured in a tissue slice from the abdominal zone of

578

D. magna pre-exposed to 40 mg-Ti/L PAA-TiO2-NPs for 24 h. Arrows indicate the location

579

of PAA-TiO2-NPs. (d) Representative energy dispersive X-ray spectrum of the PAA-TiO2-NP

580

-containing spots in (c).

581

Figure 4. The proportion of PAA-TiO2-NPs retained in Daphnia magna pre-exposed to 4

582

mg-Ti/L PAA-TiO2-NPs at a [Ca]dis of 0.2 mM for 1 h during a 48-h depuration period (Ctrl).

583

In the other two treatments, either the fleas were heat killed before the depuration (Heat) or

584

2.0 mM EGTA was added to the efflux medium (EGTA). Data are the mean ± standard

585

deviation (n = 3).

586

Figure 5. (a) The rate of PAA-TiO2-NP uptake by Daphnia magna in medium containing 0,

587

0.5, or 2.0 mM Ca with (EGTA) or without (None) 2.0 mM EGTA. EDTA (2.0 mM) was also

588

added to the treatment without Ca. (b) The rate of PAA-TiO2-NP uptake by D. magna in 21



589

medium containing 0, 0.2, or 2.0 mM Ca and with an ambient pH value of 6, 7, 8, or 9. (c)

590

The rate of PAA-TiO2-NP uptake by D. magna pre-exposed to no inhibitors (Ctrl), 1.0 mM

591

nifedipine (Nif), or 3.0 mM amiloride (Ami) in uptake medium containing 0, 0.5, or 2.0 mM

592

Ca. (d) The rate of PAA-TiO2-NP uptake by D. magna pre-exposed to 0, 1, and 10 µM BAY

593

K8644 in uptake medium containing 0, 0.2, or 2.0 mM Ca. The values of the bars with

594

different letters on the top are statistically significant (p < 0.05) from each other. Data are the

595

mean ± standard deviation (n = 3).

596

Figure 6. (a) The rate of PAA-TiO2-NP uptake by Daphnia magna pre-cultured in medium

597

containing 12.5 µM, 0.5 mM, or 5.0 mM Ca for 24 and 48 h. (b) The rate of PAA-TiO2-NP

598

uptake by D. magna in the presence of 0, 0.01, 0.04, and 0.2 µM (A–D) Ca ionophore (Ionop)

599

with a [Ca]dis of 0.2 mM. Alternatively, D. magna was pre-exposed to different concentrations

600

of thapsigargin (Thap, 0, 0.3, 1.0, and 3.0 µM for A–D) or BAPTA-AM (BAPTA, 0, 0.03,

601

0.2, and 1.0 mM for A–D) for 1 h before the accumulation of PAA-TiO2-NPs in the uptake

602

media alone, without added Ca, thapsigargin, or BAPTA-AM. Data are the mean ± standard

603

deviation (n = 3).

604

22


Page 22 of 28

Page 23 of 28

Figure 1 [Ti]daphnia (mg-Ti/g-dw)

605


10

0

PAA-TiO2-NP uptake rate (mg-Ti/g-dw/h)

0

606 607

a

2 2-re

0.2 0.2-re

20

10

20 30 40 Time (h)

50

2 b 1

ab

ab ab

a 0

0

0.2 0.5

1

2

[Ca]dis (mM)

23


b


608

Page 24 of 28

Figure 2

Ca

Ti

GT

a

0

b Ca

Ti

GT

c

0

d

AD

0

Ca

Ti

GT

e

f

609 610

24


0

Page 25 of 28

611


Figure 3

a

b

5 µm

c

d

C 400 Intensity

O Na 200 Ti

Cl 0 0

612 613 614

1

2 3 Energy (keV)

25


4

5


Figure 4 PAA-TiO2-NPs retained (%)

615

616

100

10 Heat EGTA Ctrl

1 0

10

20

30

40

Depuration time (h)

617

26


50

Page 26 of 28

Page 27 of 28

618


Figure 5


4

None EGTA EDTA

a

1.0 a

2

1.5

a a

0.5

0

3

0.5 Ctrl Nif Ami

2

0.0

2.0

c a a

ab b

0

0.2

ab

c

0.5 [Ca]dis (mM)

a a

0

2.0

a

2.0

d

a

1 a

a

0 µM 1 µM 10 µM

2

1 0

0

3

a

b

b

a

a

a

b

a a a

0

pH=6 pH=7 c bc pH=8 pH=9 aaa b a

b

a a a

a

a

0

0.2 [Ca]dis (mM)

619 620 621 622

27


2.0


623

Figure 6


1.0

a

12.5 µM 0.5 mM 5 mM

.5

0.0 1.5

24h

48h

A B

C D

b

1.0 .5 0.0

624

Page 28 of 28

Ionop

Thap BAPTA Treatments

625 626

28


TiO2 Nanoparticle Uptake by the Water Flea Daphnia magna via

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