The effects of nanomaterials in the physiology and ecology of the freshwater crustacean Daphnia ...

282 Pages·2015·4.93 MB·English

by  Kai Paul

The effects of nanomaterials in the physiology and ecology of the
freshwater crustacean Daphnia magna

Kai Benjamin Paul

Submitted for the degree of Doctor of Philosophy

Heriot-Watt University

School of Life Sciences

November 2015

The copyright in this thesis is owned by the author. Any quotation from the thesis or use
of any of the information contained in it must acknowledge this thesis as the source of
the quotation or information.
Abstract
This research studied 3 differently coated silver nanomaterials (Ag NMs) and their ionic
counterpart, dissolved Ag, focussing on their fate and toxicity in the aquatic
environment. Silver is one of the most used nanomaterials with Ag NMs degradation
potentially resulting in ionic silver release, which is well known to be highly toxic to
aquatic organisms. The organism studied here was the aquatic model species, Daphnia
magna, chosen for their non-selective particle feeding behaviour, sensitivity and
importance in the aquatic food chain (Chapter 1).
In Chapter 3 the Ag NMs physicochemical properties were characterised within the
appropriate aquatic matrices (i.e. Mill-Q water and environmental medium) using DLS,
TEM-EDX, hyperspectral imaging, UV/Vis spectroscopy, centrifugal filtration and ICP-
MS. Hyperspectral imaging was a novel technique but, highlighted is its potential
usefulness within this discipline.
Chapter 4 explores the acute and chronic toxicity of Ag NMs and dissolved Ag to D.
magna. The research here is one of the first to demonstrate that direct conversions of
acute data for chronic predictions of hazard in D. magna may not be possible and that
Ag NM toxicity cannot be fully explained by Ag+ release.
Critical modelling values were derived (biotic ligand modelling, BLM and biodynamic
model, BDM) and the best predictors of toxicity established for use in silico models
(Chapter 5).
Using biochemical techniques (Chapter 6) it was shown that Ag+ and Ag NM toxicity
differed in their modes of action (MOA). The study is the first to derive possible
schematic Ag NM MOA using multiple biochemical endpoints, with Ag+ affecting body
cations and all Ag NMs causing perturbations to mitochondrial function and oxidative
stress levels.
Within this thesis there are the first SEM images of Ag NMs trapped within the
organism filtering apparatus. The study is the first to take an in depth look at ligand
binding characteristics as they concern to Ag NMs and highlight any differences with
Ag+. In conclusion; Ag NMs are currently still one of the most toxic of all studied
NMs current levels in the environment may not cause immediate impact on D. magna
populations. However sub-lethal and long term effects under continuous exposure
may still be of concern.
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Acknowledgements
I would like to thank my supervisors Prof. Teresa Fernandes and Prof. Vicki Stone
firstly for giving me the opportunity to partake in the project and Ph.D, and secondly for
their ongoing support, kind words and encouragement as well as the invaluable insights
and expertise they gave. Not only this, but also creating opportunities and experiences
which I will never forget, and those which were key to my success. I would truly not
have been the researcher and scientist I am today without you help and guidance.
I would also like to thank the two post-docs I was lucky enough to have on the project
toward the latter stages of my Ph.D. Dr. Farhan Khan was indispensable to my
development within the body burden of toxicants and modelling arena, and my Ph.D.
Thanks for being there to listen, being a friend and accelerating what I thought was
possible. Dr. Judit Kalman thank you for your patience, expertise (particularly, again in
the modelling of body burdens) and unparalleled hard work, I know the project would
not have been able to get through such a body of work at our institute without you over
the last 6 months.
It would be remise of me to not finally thank the others within Heriot-Watt University
and the NanoSafety Group. Dr. David Brown and Dr. Birgit Gaiser thank you for your
help and guidance throughout my PhD, and all our “quick chats”. Dr. John Kinross for
always making sure we had a sound working environment and making sure I always
had what I needed. Finally thanks to the entire group and those people within DB 2.65
who made my working days what they were, and the friends I have made on the way.
Thank you Clemson University ENTOX and thank you Prof. Stephen Klaine for letting
me be part of your group, for your expertise and providing exactly what I needed
without a second thought. I also have to thank those colleagues and friends I made in
my time in the United States of America. You guys were great, and I can’t thank you
enough for your time, the rides to work and help. I feel lucky to have met you all.
I would like to thank everyone for listening to my incessant ramblings and your
patience during such times and keeping me straight; it has been a pleasure.
Thank you to Penicuik rugby club for making me feel like I had a home from home, and
providing me with a “creative outlet”.
Thanks to my now fiancé Kerry for giving me the support, love and belief I needed
when times were tough, when I was stressed or down, and pulling me out the other side.
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Thanks to Jack for keeping me young and sane. I am lucky to have not only studied for
my Ph.D but also found my own little loving family along the way.
Finally thanks to my caring and loving family, for whom it doesn’t matter how far away
I get I can always feel their support. I would not have been where I am today, let alone
able to complete a Ph.D, without you all. Thanks for believing in me and always telling
me everything is possible, thanks for always being proud and being there for me no
matter what path I chose. Thank you, Mum, Dad, Karina, Senara and my Grandparents
for always believing in me, loving and caring.
To all, my achievements and successes are yours as well as my own.
Ehaz ha sowenath whath tho why ha tho goz henath, Onen hag Oll.

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ACADEMIC REGISTRY
Research Thesis Submission

Name: Kai Benjamin Paul
School/PGI: School of Life Sciences
Version: (i.e. First, Final Degree Sought Ph.D
Resubmission, Final) (Award and
Subject area)

Declaration

In accordance with the appropriate regulations I hereby submit my thesis and I declare that:

1) the thesis embodies the results of my own work and has been composed by myself
2) where appropriate, I have made acknowledgement of the work of others and have made
reference to work carried out in collaboration with other persons
3) the thesis is the correct version of the thesis for submission and is the same version as
any electronic versions submitted*.
4) my thesis for the award referred to, deposited in the Heriot-Watt University Library, should
be made available for loan or photocopying and be available via the Institutional
Repository, subject to such conditions as the Librarian may require
5) I understand that as a student of the University I am required to abide by the Regulations
of the University and to conform to its discipline.

* Please note that it is the responsibility of the candidate to ensure that the correct version
of the thesis is submitted.

Signature of Date: 10/2015
Candidate:

Submission

Submitted By (name in capitals): KAI B. PAUL

Signature of Individual Submitting:

Date Submitted:

For Completion in the Student Service Centre (SSC)

Received in the SSC by (name in
capitals):
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Table of contents
Chapter 1 Introduction……………………………………….…………………….1-22
1.1 NanoBEE project aims and vision………………………………………..2-3
1.2 Nanomaterials and manufactured nanomaterials in the environment…...3-10
1.3 Daphnia magna.......................................................................................10-14
1.4 Toxicity of Silver Nanoparticles with focus on the Aquatic
Environment…………………………………………………………....14-18
1.5 Toxicological Modelling within the Environment ………………….....18-21
1.6 Aims……………………………………………………………………21-22
Chapter 2 Generic methodologies for nanomaterial testing, and organism testing,
culturing and maintenance……………………….……………………………….23-28
2.1 Daphnia magna culturing and testing conditions……………………....23-25
2.2 Nanomaterials used……………………………………………………..25-26
2.3 Nanoparticle synthesis and stocks ………………………………….......26-27
2.4 Statistical analyses……………………………………………………....27-28
Chapter 3 Characterisation of manufactured Silver Nanoparticles…………...29-57
3.1 Introduction……………………………………………………………..29-33
3.2 Aims……………………………………………..…………………...…33-34
3.3 Methods………………………………………………………………...34-36
3.3.1 Nanoparticles, synthesis and stocks……………………………...34
3.3.2 Characterisation ………………………………………………34-36
3.3.3 Statistical Analysis…………………………………………..…...36
3.4 Results………………………………………………………….…….....36-51
3.5 Discussion……………………………………………………..….….....51-57
3.5.1 Size, agglomeration and aggregation……………………........52-55
3.5.2 Zeta-potential……………………………………………………..55
3.5.3Dissolution…………………………………………………….......56
3.6 Conclusion………………………………………………………………….57

Chapter 4 Silver nanoparticle and silver nitrate acute and chronic toxicity to
Daphnia magna: implications for risk assessment and future
research……………………………………………………………………….……57-87
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4.1 Introduction…………………………………………………………......57-60
4.2 Aims………………………………………………………………………...60
4.3 Methods……………………………………………………………........60-63
4.3.1 Organisms…………………………………………………….......60
4.3.2 Nanoparticle synthesis and stocks……………………………......60
4.3.3 Characterisation…………………………………………………..61
4.3.4 Toxicity tests………………………………………………….61-62
4.3.5 Imaging …………………………………………………….....62-63
4.3.6 Statistical analysis……………………………………………..….63
4.4 Results………………………………………………………………......64-77
4.4.1 Toxicity tests………………………………………………….64-72
4.4.2 Imaging…………………………………………………...…..72-74
4.5 Discussion……………………………………………………………....77-86
4.5.1 Particle size and toxicity………………………………….......79-81
4.5.2 Zeta-potential and toxicity…..…………………………………....81
4.5.3 Dissolution and toxicity……………………………………….81-82
4.5.4 Potential toxicological modes of action…………………........82-86
4.6 Conclusion…………………………………………………………........86-87

Chapter 5 The Application and suitability of biodynamic and biotic ligand
modelling in silver nanoparticle and silver nitrate toxicity
assessment…………………………………………………………………...……87-115
5.1 Introduction……………………………………………………………..87-91
5.2 Aims…………………………………………………………………….91-92
5.3 Methods ………………………………………………………….……..92-98
5.3.1 Organisms………………………………………………………...92
5.3.2 Nanoparticle synthesis and stocks………………………...….…..92
5.3.3 Characterisation ………………………………………….............92
5.3.4 Toxicity tests………………………………………………...…....92
5.3.5 Waterborne exposure: uptake and efflux ………………...............93
5.3.6 Foodborne exposure: assimilation efficiency and efflux...……….94
5.3.7 Sample analysis………………………………………………......95
5.3.8 Biodynamic modelling and membrane transporter
characteristics……………………………………….………............95-97
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5.3.9 Statistical analysis………………………………………….....97-98
5.4 Results……………………………………………….………………...98-108
5.4.1 Toxicity tests…………………………………………………......98
5.4.2 Waterborne uptake of silver nanoparticles and silver nitrate.
……………………………………………………………………....98-99
5.4.3 Loss of waterborne accumulated silver nanoparticles and silver
nitrate……………………………………………………………..100-101
5.4.4 Relationship between the waterborne modelled parameters and
waterborne silver nanoparticles and silver nitrate toxicity.............101-103
5.4.5 Foodborne silver nanoparticle and silver nitrate loss dynamics and
assimilation efficiencies………………………………………….103-106
5.4.6 Biodynamic modelling of silver nanoparticle and silver nitrate
burdens from food and water in a representative freshwater
environment....……………………………………………............106-108
5.5 Discussion……………………………………………………………108-113
5.6 Conclusion…………………………………………….………...........113-115
Chapter 6 Molecular mechanisms of silver nanoparticle and silver nitrate
toxicity……………………………………………………………………...........115-158
6.1 Introduction…………………………………………………..………115-126
6.1.1 Ionoregulation in response to silver nanoparticle and silver nitrate
exposure………………………………………………….....................118
6.1.2 Energy metabolism & antioxidant defences in response to silver
nanoparticles and silver nitrate exposure………………...............119-126
6.1.2.1 Adenosine triphosphate (ATP)…………...............119-121
6.1.2.2 Lactate dehydrogenase (LDH)…………………...121-123
6.1.2.3 Mitochondria……………………………..............123-124
6.1.2.4 Oxidative stress…………………………..............124-126
6.2 Aims…………………………………………………………...…………..127
6.3 Methods………………………………………………………............127-134
6.3.1 Organisms…………………………………………..............127-128
6.3.2 Nanoparticle synthesis and stocks……………………………....128
6.3.3 Characterisation………………………………………………....128
6.3.4 Ionoregulation assessment………………………………….128-129
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6.3.5 Adenosine triphosphate (ATP), Super Oxide Dismutase (SOD) and
Catalase (CAT) extraction…………………………………….........…129
6.3.6 Adenosine triphosphate (ATP) assay……………................129-130
6.3.7 Lactate dehydrogenase (LDH) assay…………….................130-131
6.3.8 Catalase (CAT) assay……………………………………....131-132
6.3.9 Super oxide dismutase (SOD) assay…………………………….132
6.3.10 Mitochondrial membrane potential (MMP)
assessment………………………………………………………..132-133
6.3.11 Protein analysis………………………………………….……..133
6.3.12 Statistical analysis……………………………..…..……...133-134
6.4 Results……………………………………………………………......134-144
6.4.1 Ionoregulation assessment……………………….................134-136
6.4.2 Adenosine triphosphate (ATP) assay……………………....136-137
6.4.3 Lactate dehydrogenase (LDH) assay……...………………..138-140
6.4.4 Super oxide dismutase (SOD) and catalase (CAT)
assay………………………………………………….…………...140-142
6.4.5 Mitochondrial Membrane potential (MMP)
assessment……………………………………………..................142-144
6.5 Discussion…………………………………………………………....144-155
6.5.1 Ionoregualtion…………………………………...................144-147
6.5.2 Adenosine triphosphate (ATP)……………………………..147-148
6.5.3 Lactate dehydrogenase (LDH)………………......................148-150
6.5.4 Super oxide dismutase (SOD) and catalase
(CAT)……………………………………………..……….……...150-154
6.5.5 Mitochondrial membrane potential (MMP)…………..........154-155
6.6 Conclusion………………………………………………....................155-158
Chapter 7 Overall Discussion……………………………………......................158-174
7.1 Overall discussion introduction……………………………………....158-172
7.2 Final conclusions from the thesis and future researh……...................172-174
Appendix A Tables & Figures ...…………………………………….................175-181
Published Paper A…………………………………………….………………...182-206
Published Paper B………………………………………………………………207-234
Thesis References…………………………………………….............................235-259
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List of Tables:
Table 1.1: Current studies conducted on the toxicity of Ag NPs to Daphnia magna and
persisting knowledge gaps.
Table 2.1: Standard endpoints measured during D. magna toxicological assessment.
Table 3.1: Popular methods used in the physicochemical characteristic determination of
nanomaterials.
Table 3.2: Mean physicochemical characteristics of pristine (as manufactured) Ag NPs
in Milli-Q water adapted from Tejemaya et al. (2012) with the addition of ζ -potential
obtained at Heriot-Watt University (Kai B. Paul) (n = 3).
Table 3.3: Physicochemical characteristics of Ag NPs in aM7 medium and Milli-Q
water physicochemical characteristics.
Table 3.4: UV/ Vis Spectra of three Ag NPs from traditional methods (i.e. UV/ vis
spectrophotometer; abosorbance λ) at 0 and 24 hours in aM7 medium at 0 and 24 hours.
Table 4.1: Acute EC of Daphnia magna in varying silver exposures conducted on <
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24hour old neonates and 5-6 day old juveniles.
Table 4.2: NOEC and LOEC of Daphnia magna in varying silver exposures conducted
on < 24hour old neonates and 5-6 day old juveniles.
Table 4.3 Effects of AgNO , Ag NPs and food rationing to D. magna after the 21 day D.
3
magna reproduction test.
Table 5.1: Biodynamic parameters (± 95% C.I.) metal binding characteristics (± S.E.)
and lethality for D. magna exposed to Ag NO , and PVP-, PEG- and Cit-Ag NPs.
3
Table 5.2: EC correlations (Kendall’s Tau test (W statistic)) with biodynamic and
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biotic ligand model derived parameters.
Table 5.3: Biodynamic modelling of body burdens based on environmentally relevant
total silver concentrations obtained from San Francisco Bay and Monterey Bay
(Griscom, Fisher and Luoma, 2002).
Table 6.1. List of references used to determine the common MOA/AOP of silver Ag
NPs. Full references can be found in Chapter 8
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