Bioaccumulation of CdTe Quantum Dots in a Freshwater Alga

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Article

Bioaccumulation of CdTe quantum dots in a freshwater alga Ochromonas danica: a kinetics study Ying Wang, Ai-Jun Miao, Jun Luo, Zhongbo Wei, Jun-Jie Zhu, and Liuyan Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es4017188 • Publication Date (Web): 14 Aug 2013 Downloaded from http://pubs.acs.org on August 18, 2013

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Environmental Science & Technology

Table of Contents Art

Environment

Algal cell

TGA-CdTe-QDs

Plasma membrane

[QDs]cell (a.u./cell)

Diffusion 3e+4

Macropinocytosis

Dissolution Elimination

Accumulation kinetics

2e+4 Uptake 1e+4

Elimination

0 0

50 100 Time (min)

Vacuole

Expulsion Surface modification

150

ACS Paragon Plus Environment


1 2 3 4

Bioaccumulation of CdTe quantum dots in a freshwater alga

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Ochromonas danica: a kinetics study

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Ying Wang†, Ai-Jun Miao†*, Jun Luo†, Zhong-Bo Wei†, Jun-Jie Zhu‡, Liu-Yan Yang†

9 10 11

†

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Nanjing University, Nanjing, Jiangsu Province, 210046

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‡

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Province, 210046

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

School of Chemistry & Chemical Engineering, Nanjing University, Nanjing, Jiangsu

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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: The bioaccumulation kinetics of thioglycolic acid stabilized CdTe quantum

24

dots (TGA-CdTe-QDs) in a freshwater alga Ochromonas danica was comprehensively

25

investigated. Their photoluminescence (PL) was determined by flow cytometry. Its cellular

26

intensity increased hyperbolically with exposure time suggesting real internalization of

27

TGA-CdTe-QDs. This hypothesis was evidenced by the nanoparticle uptake experiment with

28

heat-killed or cold-treated cells and by their localization in the vacuoles. TGA-CdTe-QD

29

accumulation could further be well simulated by a biokinetic model used previously for

30

conventional pollutants. Moreover, macropinocytosis was the main route for their

31

internalization. As limited by their diffusion from the bulk medium to the cell surface,

32

TGA-CdTe-QD uptake rate increased proportionally with their ambient concentration. Quick

33

elimination in the PL of cellular TGA-CdTe-QDs was also observed. Such diminishment

34

resulted mainly from their surface modification by vacuolar biomolecules, considering that

35

these nanoparticles remained mostly undissolved and their expulsion out of the cells was slow.

36

Despite the significant uptake of TGA-CdTe-QDs, they had no direct acute effects on O.

37

danica. Overall, the above research shed new light on nanoparticle bioaccumulation study

38

and would further improve our understanding about their environmental behavior, effects and

39

fate.

40 41

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42

INTRODUCTION

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Engineered nanoparticles are defined as particles with size in the range of 1-100 nm in at

44

least two dimensions.1 They are widely used in various areas like medicine, electronics,

45

textile, and environmental remediation. With the ever-increasing development of

46

nanotechnology, a large quantity of nanoparticles will be released inevitably and find their

47

way into the aquatic environment yet their potential risks remain unclear. In such ecosystems,

48

algae with cell size in micron scale are often the dominant primary producers and serve as an

49

important food source for organisms at higher trophic levels. Potential nanoparticle impacts

50

on these autotrophs will not only affect the biological community composition of the aquatic

51

ecosystems but also determine the environmental fate of nanoparticles themselves. Therefore,

52

the algal toxicity of nanoparticles has been extensively investigated in recent years.2-5 Both

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indirect and direct effects were reported. Namely, nanoparticles could either liberate toxicants

54

(e.g., metal ions or reactive oxygen species) in the bulk medium and suppress the algal

55

growth indirectly or enter the cells and then impose toxic effects directly. In the latter case,

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the nanoparticles’ capability to accumulate in algae (i.e., bioavailability) determines their

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behavior, effects and fate in the natural environment. However, this property was rarely

58

examined for nanoparticles due to the lack of simple, instantaneous, and non-destructive

59

techniques to quantify their concentration in algae and other aquatic organisms. By contrast,

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the bioavailability and accumulation kinetics of heavy metals and organic pollutants have

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been well studied. Various models were established to elucidate how the accumulation

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processes are influenced by different biological and physicochemical factors.6-8 Nevertheless,

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it remains unknown whether the methodology used in these studies of conventional pollutants

64

could be applied to nanoparticles.

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In these perspectives, we investigated the bioaccumulation kinetics of quantum dots

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(QDs) in a freshwater alga Ochromonas danica. These nanoparticles are extremely small (
2 µm). Their PL inside the

260

vacuoles also intensified with time indicating a continuous uptake of these nanoparticles as

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the intracellular PL of each particle was substantially weakened which would be discussed

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below.

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Bioaccumulation of organic pollutants and heavy metals had been extensively studied in

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the past few decades. Various models were established on the uptake, elimination, trophic

265

and maternal transfer of these conventional pollutants.6,7 However, such research area is still

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in its infancy for emerging pollutants like nanoparticles. Therefore, a biodynamic model6,8

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derived from the ecotoxicological studies of conventional pollutants was adopted herein to

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validate their applicability in nanotoxicology. According to this model, the variation of

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[QDs]intra with time in the uptake experiment could be illustrated by the equation below,

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d[QDs]intra = k u × [QDs]med − kem × [QDs]intra dt 9


(1)

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where ku (ml/cell/min) is the uptake rate constant (also called clearance rate). [QDs]med

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(a.u./ml) means the PL-based concentration of TGA-CdTe-QDs in the uptake medium and

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the nanoparticle uptake rate could thus be expressed as ku×[QDs]med (a.u./cell/min). Further,

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kem (min-1) represents the elimination rate constant for the intracellular PL of

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TGA-CdTe-QDs including those expelled out of the cells (kex, min-1) and those quenched

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inside as a result of surface modification or dissolution. On the other hand, bioaccumulation

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could reduce [QDs]med, which may partly be compensated by their expulsion afterwards as

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follows,

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d[QDs] med = ( − k u × [QDs] med + k ex × [QDs]intra ) × d dt

(2)

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where d (cells/ml) means cell density. As TGA-CdTe-QD expulsion out of the cells was

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rather slow (discussed later), its contribution (i.e., kex × [QDs]intra × d ) to the variation of

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[QDs]med could be neglected. According to Eqs. (1) and (2), [QDs]intra could be derived, 0

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[QDs]intra =

ku × [QDs]med × (e −ku ×d ×t − e −kem×t ) kem − ku × d

(3)

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where [QDs]med0 (a.u./ml) signifies the initial PL-based concentration of TGA-CdTe-QDs in

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the uptake medium after quick surface adsorption. Further,

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[QDs]cell = [QDs]intra + [QDs]ads

(4)

Therefore, 0

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[QDs]cell =

k u × [QDs]med × (e −ku ×d ×t − e −kem ×t ) + [QDs]ads k em − k u × d

(5)

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If [QDs]med remained unchanged (< 20%) during the uptake period as was the case in the

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present study, Eq. (5) can be further simplified, 0

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[QDs]cell

k × [QDs]med = u × (1 − e −kem ×t ) + [QDs]ads k em

(6)

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Eq. (6) was then used to fit the hyperbolic correlation between [QDs]cell and uptake duration.

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The two constants ku and kem ranged from 2.15×10-9 to 4.92×10-9 ml/cell/min and from 0.018

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to 0.062 min-1 with no consistent trend between the different concentration treatments. Wu et

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al.29 quantified the clearance rate of Ochromonas sp. for its predation of various filamentous

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bacteria. It was in the range of 1.15-3.13×10-8 ml/cell/min, which was approximately one 10



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order of magnitude higher than what we found here. One explanation is the difference in their

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prey size (1.1-13.7 µm for bacteria vs. 88.9 nm for TGA-CdTe-QDs) which determines the

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clearance rate and uptake routes (phagocytosis vs. pinocytosis).30-32 Further, TGA-CdTe-QD

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uptake rate increased linearly with [QDs]med (Figure 1d), suggesting the internalization

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processes were way below saturation. Considering their remarkable dissolution, the actual

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concentration of TGA-CdTe-QDs in different uptake media was 3.20, 1.95, 1.31, 1.03, 0.83,

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0.65 mg-Cd/l, as measured by GFAAS after ultrafiltration through a 10 kD membrane.

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According to their hydrodynamic size in DY-V and the number of Cd for each primary

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particle, TGA-CdTe-QD uptake rate in the unit of aggregates/cell/min was approximately

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13.3 - 61.2 which was more rapid than the bacteria ingestion rate (0.1-0.5 bacteria/cell/min)

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of Ochromonas sp..29 Such discrepancy was mainly due to much higher particle concentration

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in the former case. If we assume that the migration vesicles with diameter approximately 1

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µm as shown in Figure 2 were filled with TGA-CdTe-QD aggregates, then it could be

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estimated that 0.015-0.067% of the plasma membrane was invaginated every minute for each

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single cell. It was much lower than that of macrophages which can ingest 3% of its plasma

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membrane every minute.33 The ciliate amoebae have an even faster membrane recycling rate

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than macrophages suggesting their different internalization pathway or the unsuitability of

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TGA-CdTe-QDs to be taken up by O. danica.

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Cellular uptake of nutrients or pollutants (including nanoparticles) generally includes

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three steps, 1) diffusion from the bulk solution to the cell periphery; 2) surface

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adsorption/binding; 3) internalization.34 Considering our TGA-CdTe-QDs as rigid spheres,

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their diffusion coefficient (D, cm2/s) at infinite dilution could be calculated using the

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Stokes-Einstein relationship,

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D=

k BT 6πηs RH

(7)

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where kB (cm2 kg/s-2/K-1) is the Boltzmann constant, T (K) and ηs (kg/s/m) signify absolute

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temperature and solvent viscosity, RH (m) is the hydrodynamic radius of the particle as

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determined by DLS. The diffusion coefficient of TGA-CdTe-QDs in DY-V was thus 4.82 ×

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10-8 cm2/s. Then their maximum diffusion to the cell surface Jmax (a.u./cell/min) could be

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calculated by the equation below,34

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J max = 60 D × S × ([QDs]med − [QDs]i ) × (

1 1 + ) R δ

11


(8)

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where S (cm2/cell) indicates the surface area of each cell, [QDs]i (a.u./ml) is the nanoparticle

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concentration in the interface between cells and the bulk solution, R (4×10-4 cm) and δ (cm)

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signify the radius of O. danica and the diffusion layer thickness. Assuming that

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TGA-CdTe-QD uptake by the cells is rather quick (i.e., [QDs]i = 0 a.u./ml) and δ is much

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bigger than R,34 then Jmax could be calculated to be in the range of 10.5-3076.1 a.u./cell/min.

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Their actual uptake rate (2.3-637.2 a.u./cell/min) was 14.8-33.9% of the maximum diffusion.

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Therefore, TGA-CdTe-QD diffusion from the bulk solution to the cell surface determined

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their internalization by O. danica.35 It could further be anticipated that nanoparticle

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bioaccumulation may be restricted by their physical transport unless their hydrodynamic size

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is below 10 nm. This hypothesis was supported by the study of Chen et al.36, in which uptake

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of “colloidal iron” (~ 5 nm) by the marine diatom Thalassiosira pseudonana was investigated.

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Its internalization was 0.08-0.2% of the maximum diffusive fluxes and thus was not limited

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by diffusion.

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Uptake and Elimination Mechanisms. Endocytosis has been well-recognized to be the

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major mechanism for nanoparticles to enter cells.31 Two main categories, phagocytosis and

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pinocytosis, are included and the latter is further classified into at least four mechanisms (i.e.,

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clathrin-mediated,

caveolae-mediated,

clathrin/caveolae-independent

endocytosis, 30,37

and

344

macropinocytosis) depending on their formation of intracellular vesicles.

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experiment of the present study, the mixotrophic freshwater alga O. danica was growing

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quite well excluding the possibility that TGA-CdTe-QDs entered the cells by altering their

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plasma membrane permeability.4 This alga is also well-known for its ability to ingest bacteria

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and yeasts (i.e., “cell eating”) through phagocytosis,15 which is mainly employed to take up

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particles larger than 0.5 µm.30 Therefore, this mechanism may not be applicable to the

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nano-sized particles, which would be corroborated by the following finding. To gain further

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insight into the specific mechanism for the translocation of TGA-CdTe-QDs into O. danica,

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six inhibitors were applied. Among them, CCCP and NaN3 are able to impair ATP production

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by uncoupling oxidative phosphorylation38 or impeding cytochrome oxidation.39 The

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presence of both inhibitors resulted in a marked decline of TGA-CdTe-QD uptake rate (Table

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1), indicating their internalization is an energy-consuming process. MβCD is a cyclic

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heptasaccharide and could deplete cholesterol or modify cholesterol-rich domains (lipid rafts)

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within the cell membrane.40 It is thus often used as a selective inhibitor of caveolae-mediated

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uptake.41 But its suppression of macropinocytosis and clathrin-dependent endocytosis was

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also reported.42,43 By contrast, genistein is a depressor of several tyrosine kinases and could

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mainly restrain the caveolae- and clathrin-mediated endocytosis.44 Further, dynasore impedes 12


In the uptake


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the function of dynamin and this protein is required in various endocytosis processes (e.g.,

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phagocytosis) but not including macropinocytosis.30 The latter pathway could however be

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blocked by amiloride due to the reduction of submembraneous pH together with the

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suppression of Rac1 and Cdc42 signaling.45 As shown in Table 1, a substantial decrease in

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TGA-CdTe-QD uptake was observed when MβCD or amiloride was applied, which was

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further aggravated at higher concentrations of these two inhibitors. On the contrary, both

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genistein and dynasore had no significant effects (p > 0.05). Moreover, the vacuoles

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encapsulating TGA-CdTe-QDs inside the cells (> 1 µm, Figure 2) were much larger than

369

those involved in various pinocytosis processes (~ 100 nm, Table 1) other than

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macropinocytosis.31,46 In viewed of all these phenomena, macropinocytosis was thus

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considered to be the main route for TGA-CdTe-QDs to be taken up by O. danica. Similarly,

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this mechanism was previously found to be responsible for the uptake of peptide-conjugated

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QDs by HeLa cancer cells.47 Nevertheless, QDs could still be endocytosed via other routes

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like clathrin-mediated pinocytosis and sometimes their bioaccumulation may even be

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accomplished through multiple pathways collectively.48 Further research is required to

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illuminate how the internalization routes are selected based on the characteristics of

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nanoparticles or cells themselves.

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[QDs]cell (a.u./cell) represents the cellular concentration of TGA-CdTe-QDs based on

379

their PL. Therefore, the hyperbolic correlation between [QDs]cell and exposure time in the

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uptake experiment was speculated to be caused by the quick elimination of TGA-CdTe-QD

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PL intracellularly as a result of their dissolution, surface modification, or expulsion out of the

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cells. The intracellular diminishment of TGA-CdTe-QD PL was supported by the finding that

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[QDs]cell decreased by more than 90% within a few hours after the ambient nanoparticles

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were removed in the efflux experiment (Figure 3a). Moreover, [QDs]cell went up again once

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the cells were re-exposed to TGA-CdTe-QDs, excluding the possibility that [QDs]cell leveled

386

off due to the physiological changes of O. danica in the uptake experiment. On the other hand,

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no PL signal of TGA-CdTe-QDs was found in the efflux medium and the cellular contents of

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Cd or Te decreased by no more than 10% after 4.5 h (Figure 3b). It implies that

389

TGA-CdTe-QD expulsion out of the cells either in the form of nanoparticles or metal ions

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was relatively slow and intracellular PL quenching was the main cause for the substantial

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diminishment of [QDs]cell. The exocytosis/expulsion of single-walled carbon nanotubes and

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gold nanoparticles by the fibroblast NIH-3T3 cells were examined with the efflux rate

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constant (i.e., kex) in the range of 10-4 to 10-3 min-1.49 In other words, approximately

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2.4-21.3% of the intracellular nanoparticles were expelled out during a 4.5-h period, 13


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comparable to what was estimated based on the variation of cellular metal contents in the

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efflux experiment of the present study. The decrease of [QDs]cell with time was then

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simulated by the first order kinetics below,

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[QDs]cell = [QDs]cell × e - kem ×t

0

(9)

399

where [QDs]cell0 (a.u./cell) is the cellular concentration of TGA-CdTe-QDs at the beginning

400

of the efflux experiment. The elimination rate constant kem thus obtained was 0.057 min-1 as

401

was similar to its counterpart (0.062 min-1) in the uptake experiment. Then was the

402

intracellular PL quenching because of TGA-CdTe-QD surface modification or their

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dissolution in the acidic vacuoles?31,50 To answer this question, the PL of TGA-CdTe-QDs in

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the cell homogenate of O. danica as a simulation of the intracellular environment was

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monitored. It diminished by more than 90% within 10 min while the wavelength of their PL

406

maximum remained unchanged as in contrast to what was found in Domingos et al.51. In this

407

situation, the characteristic XRD peaks of CdTe still existed with similar height to the control

408

treatment (Figure 3c) where the same proportion of TGA-CdTe-QDs and cell homogenate

409

were freeze-dried before being mixed with each other (i.e., possible dissolution of the

410

nanoparticles was avoided). Additionally, our preliminary experiment illustrated that the PL

411

of TGA-CdTe-QDs remained constant with the ambient pH in the range of 4-8, excluding the

412

potential effects of vacuolar acidic environment on PL quenching. Therefore, it is mainly

413

TGA-CdTe-QD surface modification due to the existence of biomolecules (e.g., amino acids,

414

proteins, carbohydrates and lipids etc.) in the vacuoles which eliminated their PL abruptly.

415

Likewise, nanoparticles in biological fluids tend to be coated by proteins which was also

416

named as protein corona.52 Furthermore, an irreversible quenching of QD PL by DNA,

417

nucleotides, amino acids was reported,53 which may result from the redox reaction between

418

the QDs and biomolecules or the binding of the quenchers to the nanoparticle surface via

419

electrostatic attraction, hydrogen bonding and so on.

420

Toxicity of TGA-CdTe-QDs. Metallic nanoparticles may have either direct or indirect

421

adverse effects on algae depending on their ability to enter cells.2,16 In the literature, most of

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the algal toxicity of nanoparticles was attributed to their indirect effects with relatively few

423

observations of direct ones.4,5,16 Silver nanoparticles could get into the cells of the same algal

424

species as what was used herein and were concentrated also in vacuoles.16 Further, these

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nanoparticles were noxious even when the ambient free silver ion concentration was orders of

426

magnitude lower than the non-observed effect level, suggesting that silver nanoparticles

427

inside the cells did have some direct effects. Similarly, natural organic matter was able to 14



428

induce the uptake of CuO nanoparticles with size smaller than 5 nm by a prokaryotic alga

429

Microcystis aeruginosa and thus increase their nanotoxicity.54 In the present study, a large

430

quantity of TGA-CdTe-QDs was internalized by O. danica. However, their adverse effects

431

were comparable to what was found in the < 10 kD ultrafiltrate of these nanoparticles with

432

similar [Cd]dis (Figure 4). Accordingly, the relative change of µ with or without the presence

433

of TGA-CdTe-QDs could be fitted to the same dose-response curve. Although Te was also

434

present in the ultrafiltrate, its concentration was not high enough to be toxic based on the

435

inhibition results of TeO32- (SI, Figure S5), the most noxious form of Te.55 Therefore, our

436

findings revealed that the intracellular TGA-CdTe-QDs had negligibly direct acute effects on

437

O. danica and their toxicity was mainly caused by Cd ion liberation into the bulk medium.

438

Possible explanations include the disposition of the nanoparticles in relatively inert

439

compartments (e.g., vacuoles). Surface modification by intracellular biomolecules may also

440

alleviate their toxicity.56 Kirchner et al.57 found that the Cd released from intracellular QDs

441

were more toxic than those dissolved outside. It implies that most of TGA-CdTe-QDs

442

remained undissolved in O. danica further supporting our hypothesis that intracellular PL

443

quenching was mainly brought about by nanoparticle modification on the surface instead of

444

dissolution.

445

Overall, our study manifests that TGA-CdTe-QDs could enter the freshwater alga O.

446

danica directly through macropinocytosis. Once they were in the cells, these nanoparticles

447

were concentrated in vacuoles with limited exocytosis/expulsion and dissolution, resulting in

448

negligible toxicity to the alga. Moreover, the PL of intracellular TGA-CdTe-QDs got

449

eliminated rather quickly as a result of their surface modification. Such instability of PL

450

inside the cells cannot be disregarded when cell-targeting QDs with new functions are to be

451

synthesized in the future. More importantly, a biodynamic model previously used for

452

conventional pollutants was for the first time systematically applied herein to illustrate

453

nanoparticle bioaccumulation kinetics in aquatic organisms, thanks to the PL of QDs. The

454

methodology thus established might be extended to other environmental studies using

455

different organisms in various environments as long as the objective nanoparticles could be

456

analyzed rather simply, instantaneously, and non-destructively (e.g., fluorescence or isotope

457

labeling).

458

ACKNOWLEDGEMENTS

459

We thank two anonymous reviewers for their constructive suggestions on this paper. The

460

financial supports offered by the National Natural Science Foundation of China (41001338, 15


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41271486, and 21237001) and the Natural Science Foundation of Jiangsu Province

462

(BK2010371) to A. J. Miao have made this work possible.

463

SUPPORTING INFORMATION

464

Detailed procedures about nanoparticle synthesis and characterization, the PL spectra of

465

TGA-CdTe-QDs and O. danica, physicochemical characterization of TGA-CdTe-QDs,

466

nanoparticle PL stability in DY-V and verification of flow cytometer’s ability to properly

467

detect TGA-CdTe-QDs in the medium, discrimination of extracellular nanoparticles from O.

468

danica and quantification of nanoparticle bioaccumulation by flow cytometry, as well as the

469

toxicity of TeO32- are included as Supporting Information. This information is available free

470

of charge via the Internet at http://pubs.acs.org.

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630 631

Page 22 of 27

Table 1. Suppression of TGA-CdTe-QD uptake in the presence of different concentrations of inhibitors. Pinocytosis Inhibitors

NaN3 CCCP MβCD Amiloride Genistein Dynasore 632 633 634 635 636

Conc.

Inhibition (%)

10 mM

19.7*

20 mM

**

44.1

10 µM

5.45

20 µM

36.3*

50 mM

35.7*

100 mM

47.1**

0.4 mM

28.7*

0.6 mM

37.1**

0.2 mM

-26.3

0.4 mM

-6.5

1 µM

5.1

4 µM

8.8

Phagocytosis (> 1 µm)a

Macropinocytosis (> 0.5 µm)

Clathrin-mediated (~120 nm)

Caveolae-mediated (~50 – 80 nm)

Clathrin/Caveolae-independent (~90 nm)

+b

+

+

+

+

+

+

+

+

+

+

+

+

-c

-

-

-

+

-

a. b. c. * .

The size of the vesicles involved in this endocytosis route. This endocytosis route could be suppressed by the inhibitor of the same row and significant uptake inhibition was observed. This endocytosis route could be suppressed by the inhibitor of the same row but no significant uptake inhibition was observed. p < 0.05. ** . p < 0.01.

21


Page 23 of 27


637

Figure Legends

638

Figure 1. Variation of cellular TGA-CdTe-QDs ([QDs]cell, a.u./cell) with exposure time (a, b)

639

in the presence of 1.3, 1.5, 1.9, 2.3, 3.1, 4.7 mg-Cd/l nanoparticles and (c) in the uptake

640

experiment with heat-killed (heat, star) or cold-treated (cold-0, square; cold-15, triangle) cells

641

as compared to the normal uptake (circle). The arrow indicates transferring cells from 25 oC

642

to 4 oC after 15-min normal uptake. Ambient TGA-CdTe-QD concentration was kept at 4.7

643

mg-Cd/l throughout this experiment. Solid lines in (a)-(c) represent the simulated increase of

644

[QDs]cell with exposure time; (d) Linear correlation between TGA-CdTe-QD uptake rate and

645

their ambient concentration in the medium. Data are mean ± standard deviation (n = 2).

646

Figure 2. The confocal laser scanning microscopy images of O. danica as obtained via

647

different channels (CH1: differential interference contrast; CH2: 405 nm laser, filter bandpass

648

= 656-702 nm; CH3: 405 nm laser, filter bandpass = 565-610 nm; Merge: combinative

649

images from CH1 to 3) or under the z-scanning mode at a 1.24 µm spacing (30 min only)

650

after exposed to 4.7 mg-Cd/l TGA-CdTe-QDs for 0, 5, 30, and 60 min. Scale bars are 5 µm.

651

Figure 3. (a) Decrease of cellular TGA-CdTe-QDs ([QDs]cell, a.u./cell) with depuration time

652

in a 4.5-h efflux experiment. The arrow indicates a subset of the cells was exposed to 4.7

653

mg-Cd/l TGA-CdTe-QDs again after 30 min in this experiment. Solid lines represent the

654

simulated curves for the elimination of [QDs]cell and the reuptake of TGA-CdTe-QDs; (b) The

655

relative amount of Cd (circle) and Te (triangle) retained as compared to their initial cellular

656

content in the efflux experiment. Solid lines represent the relative changes of cellular metal

657

content with depuration time as simulated by the first-order kinetics; (c) XRD patterns (top)

658

for the freeze-dried mixture of TGA-CdTe-QDs and cell homogenate as well as (bottom) for

659

the control treatment where the nanoparticle solution and cell homogenate were dried

660

separately before being mixed with each other. The cell homogenate was prepared by

661

suspending the algal cells in 0.9% w/w sodium chloride before breaking them through

662

sonication. Sodium chloride also served as an internal standard for the quantification of CdTe.

663

Data are mean ± standard deviation (n = 2).

664

Figure 4. Relative changes of the cell specific growth rate µ at different dissolved

665

concentration of Cd ([Cd]dis) for Ochromonas danica exposed to different concentrations of

666

TGA-CdTe-QDs (1.4, 2.9, 5.9, 12, 32, and 62 mg-Cd/l) or their corresponding < 10 kD

667

ultrafiltrates for 24 h as compared to the control treatment. Solid line is the simulated

668

dose-response curve by the Logistic model. Data are mean ± standard deviation (n = 2). 22



2e+3

c 2e+4

a e

normal 1.9


1e+3

1e+4

cold-15 1.5

cold-0 1.3

0 0

20

40

2e+4

heat

60 0 Time (min)

20

40

60 d

b

0

600

4.7

1e+4

300

3.1 2.3

0 0 670 671 672

20 40 Time (min)

60

0

1e+11 2e+11 [QDs]med (a.u./ml)

23



Figure 1

0 3e+11

Uptake rate (a.u./cell/min)

669

Page 24 of 27

Page 25 of 27

673


Figure 2 CH 2

CH 3

Z-scanning

60 min

30 min

5 min

0 min

CH 1

674 675 676

24


Merge


Figure 3 [QDs]cell (a.u./cell)

677

a

3.0e+4

reuptake

1.5e+4 elimination

0.0 Metal retained (%)

150

b Cd

100

Te

50 0 0

100

200

300

Time (min)

678

Counts (a.u.)

1000

c CdTe

500

NaCl

0 10

30 50 2θ (degrees)

679 680 681 682

25


70

Page 26 of 27

Page 27 of 27

Figure 4

Relative change of µ

683


1.0

0.5 < 10 kD QDs

0.0 0

684

1 2 [Cd]dis (mg/l)

3

685 686 687 688 689 690

26


Bioaccumulation of CdTe Quantum Dots in a Freshwater Alga

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