Colloidal stability of silver nanoparticles and their interactions with the alga Chlamydomonas ...

109 Pages·2012·3.17 MB·English

ETH Library
Colloidal stability of silver
nanoparticles and their interactions
with the alga Chlamydomonas
reinhardtii
Doctoral Thesis
Author(s):
Piccapietra, Flavio
Publication date:
2012
Permanent link:
https://doi.org/10.3929/ethz-a-007339215
Rights / license:
In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection.
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DISS. ETH NO. 20365

Colloidal stability of silver nanoparticles and their
interactions with the alga Chlamydomonas reinhardtii

A dissertation submitted to
ETH ZURICH

for the degree of
Doctor of Sciences

Presented by

Flavio Piccapietra

Diploma in Environmental Sciences, ETH Zurich
born on July 26, 1981
Citizen of Corippo (TI)

Accepted on the recommendation of

Prof. Dr. Laura Sigg, examiner
Prof. Dr. Bernhard Wehrli, co-examiner
Prof. Dr. Pedro J. J. Alvarez, co-examiner
Dr. Renata Behra, co-examiner

2012

Table of content
Table of content
Summary ........................................................................................................................ VII
Riassunto .......................................................................................................................... IX
Zusammenfassung ........................................................................................................... XI
1. Introduction ................................................................................................................... 1
1.1 Production and application of engineered silver nanoparticles (AgNP) ........................... 1
1.2 Synthesis of AgNP ............................................................................................................ 2
1.3 Physicochemical properties of engineered nanoparticles (NP) ......................................... 3
1.4 Measurement of NP size and surface charge .................................................................... 4
1.5 Dissolution of AgNP ......................................................................................................... 5
1.6 Release of AgNP into the aquatic environment ................................................................ 6
1.7 Fate of AgNP in the aquatic environment ......................................................................... 6
1.8 Bioavailability and uptake of silver in algae ..................................................................... 8
1.9 Toxicity of AgNP to algae and bacteria ............................................................................ 9
1.10 Scope of the thesis ......................................................................................................... 10
1.11 References ..................................................................................................................... 12
2. Colloidal stability of carbonate-coated silver nanoparticles in synthetic and
natural freshwater .......................................................................................................... 19
2.1 Abstract ........................................................................................................................... 20
2.2 Introduction ..................................................................................................................... 20
2.3 Materials and methods .................................................................................................... 22
2.4 Results ............................................................................................................................. 25
2.4.1 AgNP stock suspension ..................................................................................................... 25
2.4.2 Effect of pH....................................................................................................................... 26
2.4.3 Effect of ionic strength ...................................................................................................... 28
2.4.4 Effect of humic acids ........................................................................................................ 29
2.4.5 Stability in natural water ................................................................................................... 31
2.5 Discussion ....................................................................................................................... 32
2.6 Acknowledgement ........................................................................................................... 35
2.7 References ....................................................................................................................... 36
2.8 Supporting information ................................................................................................... 38
III
Table of content
2.9 Dissolution of carbonate-coated silver nanoparticles in natural freshwater .................... 41
2.9.1 Introduction ....................................................................................................................... 41
2.9.2 Materials and methods ...................................................................................................... 41
2.9.3 Results ............................................................................................................................... 42
2.9.4 Discussion ......................................................................................................................... 43
2.9.5 References ......................................................................................................................... 44
3. Intracellular silver accumulation in Chlamydomonas reinhardtii upon exposure to
carbonate-coated silver nanoparticles and silver nitrate ............................................ 45
3.1 Abstract ........................................................................................................................... 46
3.2 Introduction ..................................................................................................................... 46
3.3 Materials and methods .................................................................................................... 48
3.4 Results ............................................................................................................................. 53
3.4.1 Nanoparticle characterization............................................................................................ 53
3.4.2 Wash experiments ............................................................................................................. 53
3.4.3 Accumulation experiments ............................................................................................... 54
3.4.4 Uptake models and kinetics .............................................................................................. 57
3.5 Discussion ....................................................................................................................... 59
3.6 Acknowledgement ........................................................................................................... 61
3.7 References ....................................................................................................................... 62
3.8 Supporting information ................................................................................................... 64
4. Determinant role of silver ions in the toxicity of silver nanoparticles to
Chlamydomonas reinhardtii ............................................................................................ 71
4.1 Abstract ........................................................................................................................... 72
4.2 Introduction ..................................................................................................................... 72
4.3 Materials and methods .................................................................................................... 74
4.4 Results ............................................................................................................................. 78
4.4.1 Nanoparticle characterization............................................................................................ 78
4.4.2 Effects on photosynthetic yield ......................................................................................... 78
4.4.3 Effects on intracellular esterase activity............................................................................ 80
4.4.4 Effects on membrane integrity .......................................................................................... 81
4.4.5 Effects on photosynthetic yield as a function of intracellular silver content ..................... 82
4.5 Discussion ....................................................................................................................... 83
4.6 Acknowledgement ........................................................................................................... 85
4.7 References ....................................................................................................................... 86
4.8 Supporting information ................................................................................................... 88
IV
Table of content
5. Outlook ......................................................................................................................... 93
5.1 Fate of AgNP in the aquatic environment ....................................................................... 93
5.2 AgNP uptake in algae ...................................................................................................... 94
5.3 Effects of AgNP to algae ................................................................................................. 95
Acknowledgements ......................................................................................................... 97

V

Summary
Summary
Information on the fate, bioavailability, and toxicity of engineered silver
nanoparticles (AgNP) is needed to evaluate risks posed by their entering in the aquatic
environment. Thus, the aim of this PhD thesis is to investigate the fate of carbonate-
coated AgNP under natural water conditions, and their bioavailability and toxicity to the
green alga Chlamydomonas reinhardtii, by discriminating between the direct effects of
the AgNP and the indirect effects induced by the free silver ions (Ag+).
To investigate the colloidal stability of AgNP, the influence of pH, ionic strength,
and humic substances on their physicochemical properties was systematically
investigated in synthetic and natural freshwater. The stability of AgNP depended on the
properties and stability of the particle coating. According to size measurements, an
increased AgNP agglomeration was measured below pH 4 and at electrolyte
concentrations above 2 mM Ca2+ and 100 mM Na+. The stability of AgNP in natural
freshwaters was also identified to be primarily controlled by the electrolyte type and
concentration. Thus, according to the chemical properties of an aquatic system, a certain
degree of mobility and persistence of slightly agglomerated AgNP can be expected.
To explore AgNP bioavailability to algae and to inform on the role of the algal cell
wall, the intracellular silver uptake upon exposure to 0.5-10 µM AgNP and 20-500 nM
AgNO was investigated in the wild type and in the cell wall free mutant of
3
C. reinhardtii. To measure intracellular concentrations, a wash procedure to remove Ag+
and AgNP from the algal cell surface was performed. The results indicated an increased
silver uptake with increasing exposure time and AgNP and AgNO concentrations in the
3
exposure media. Steady-state concentrations between 10-5 and 10-3 mol L -1 were
cell
reached after 20 to 60 minutes. In case of Ag+ and according to Michaelis-Menten
analysis, high uptake kinetics values (J , 8 x 10-9 mol m-2 min-1; V , 8 x 10-6 mol
max max
L -1 min-1; K , 10-7 mol L-1) and bioconcentration factors (> 103 L L -1) were
cell m cell
determined. Due to the higher uptake rates in the mutant, a protective role of the cell wall
in limiting Ag+ uptake was suggested. Additionally, AgNP bioavailability was calculated
to be low in both strains relative to Ag+, indicating a limited AgNP internalization across
the cell membrane.
VII
Summary
To examine the acute toxicity of AgNP, both strains of C. reinhardtii were exposed
to 0.1-500 µM AgNP and 0.01-10 µM AgNO , and their effects on photosynthesis,
3
intracellular esterase activity, and membrane integrity were measured. To discern
between the effect of the nanoparticles themselves and the effect of Ag+, experiments
were performed in the presence of the silver ligand cysteine, which upon complexation of
Ag+ abolished toxicity induced by Ag+. The results showed that for all assessed endpoints
inhibitory effects of AgNP are determined by Ag+, and that the mutant had a higher
sensitivity than the wild type. Due to the higher sensitivity of photosynthesis (EC after
50
1 hour = 18-37 nM Ag+, for the mutant) in comparison to the other endpoints (EC after
50
1 hour = 157-645 nM Ag+, average for both strains), a specific interaction of Ag+ with the
photosystem II was hypothesized.
The effects of AgNP and AgNO on photosynthesis were also described as a
3
function of intracellular silver concentrations. In both strains, the photosynthetic yield
was inhibited by similar silver concentration (EC after 1 hour = 10-4 mol Ag L -1),
50 cell
suggesting that the different sensitivity between the wild type and the mutant was induced
by the different silver uptake kinetics, and not by different mechanisms of metal toxicity
and detoxification.
In conclusion, this work showed that, under specific conditions, AgNP might be
stable and persistent in the aquatic environment. Concerning the AgNP bioavailability
and toxicity to the green alga C. reinhardtii, both intracellular uptake and effects were
observed to be mainly related to the highly bioavailable Ag+, which may be released from
AgNP by dissolution, while the direct AgNP bioavailability was low.

VIII
Riassunto
Riassunto
Lo studio sul comportamento, la disponibilità biologica, e la tossicità delle
nanoparticelle di argento (AgNP) è necessario al fine di valutare i rischi derivanti dalla
loro possibile immissione nel sistema acquatico. Pertanto, lo scopo di questa tesi é quello
di esaminare il comportamento di AgNP ricoperte di carbonato in sistemi acquatici che
mimano le principali caratteristiche delle acque naturali. La loro disponibilità biologica e
tossicità nei confronti dell’alga verde Chlamydomonas reinhardtii è stata studiata
differenziando sia gli effetti diretti delle AgNP che quelli derivanti dall’argento ionico
(Ag+).
L’analisi della stabilità colloidale delle AgNP in acque naturali e sintetiche è stata
eseguita analizzando sistematicamente gli effetti del pH, della forza ionica, e delle
sostanze umiche sulle loro proprietà fisiche e chimiche. I risultati hanno evidenziato una
maggiore agglomerazione delle AgNP a pH minori di 4 e in presenza di alte
concentrazioni di calcio (2 mM Ca2+) e sodio (100 mM Na+). Inoltre la concentrazione ed
il tipo di sali sono stati identificati come i fattori predominanti per il controllo della
stabilità delle AgNP in acque naturali. Di conseguenza, a seconda delle proprietà
chimiche di un sistema acquatico, è possibile prevedere un diverso grado di mobilità e
persistenza delle AgNP.
Al fine di esaminare la disponibilità biologica delle AgNP per le alghe e di avere
maggiori informazioni sul ruolo della loro parete cellulare, è stata anche esaminata
l’assimilazione intracellulare di argento sia nel ceppo selvatico dell’alga C. reinhardtii
che in quello mutante, privo della parete cellulare, a concentrazioni di AgNP e AgNO ,
3
rispettivamente, comprese tra 0.5-10 µM e 20-500 nM. La misura delle concentrazioni
intracellulari di argento è stata eseguita con una procedura che rimuove le AgNP e l’Ag+
dalla superfice delle alghe. Una maggiore assimilazione intracellulare è stata osservata in
funzione del tempo di esposizione e in presenza di una crescente concentrazione di AgNP
e AgNO nel mezzo di coltura. Le concentrazioni intracellulari di argento allo stato
3
stazionario (10-5 and 10-3 mol L -1) sono state raggiunte dopo un intervallo di tempo
cell
compreso tra 20 e 60 minuti di esposizione. Nel caso dell’Ag+, una veloce cinetica di
assimilazione (J , 8 x 10-9 mol m-2 min-1; V , 8 x 10-6 mol L -1 min-1; K , 10-7 mol
max max cell m
L-1) e gli elevati fattori di bioaccumulazione (> 103 L L -1) sono stati determinati
cell
IX