Investigating the effects of organic pollutants on amphibian populations in the UK

286 Pages·2016·10.65 MB·English

by  Strong, Rebecca

Investigating the effects of organic
pollutants on amphibian populations in
the UK

A thesis submitted for the degree of Doctor of
Philosophy in the Faculty of Science and Technology,
Lancaster University

Alternative format thesis

March 2016

Rebecca Jane Strong (MSc)

Lancaster Environment Centre
Abstract
Amphibians are undergoing dramatic population declines, with environmental
pollution reported as a significant factor in such declines. Technologies are required
that are able to monitor populations at risk of deteriorating environmental quality in a
rapid, high-throughput and low-cost manner. The application of biospectroscopy in
environmental monitoring represents such a scenario. Biospectroscopy is based on the
vibrations of functional groups within biological samples and may be used to
signature effects induced by chemicals in cells and tissues. Here, attenuated total
reflection Fourier-transform infrared (ATR-FTIR) spectroscopy in conjunction with
multivariate analysis was implemented in order to distinguish between embryos,
whole tadpoles at an early stage of development and individual tissues of late-stage
tadpoles of the common frog collected from ponds in the UK with varying levels of
water quality, due to contamination from both urban and agricultural sources. In
addition, a Xenopus laevis cell line was exposed to low-levels of fungicides used in
agriculture and assessed with ATR-FTIR spectroscopy. Embryos, in general did not
represent a sensitive life stage for discriminating between ponds based on their
infrared spectra. In contrast, tadpoles exposed to agricultural and urban pollutants,
both at early and late stages of development were readily distinguished on the basis of
their infrared spectra. ATR-FTIR spectroscopy also readily detected fungicide-
induced changes in X.laevis cells, both as single-agent and binary mixture effects.
Data reported in this study confirm the use of ATR-FTIR spectroscopy as a sensitive
technique capable of detecting small changes in cellular groups, and as such
represents a valuable starting point for its use in the monitoring of amphibian
populations. However further research is needed in order to overcome confounding
factors existent in natural populations of complex organisms.
II
Acknowledgements
This research would not have been possible without the assistance of many
individuals. I would like to thank my supervisors: Professor Frank Martin, Dr Crispin
Halsall, Professor Richard Shore and Professor Kevin Jones. Both Frank and Crispin
offered me guidance and support throughout my PhD for which I am extremely
grateful.
Lab A20 welcomed me from the start, providing me with training and friendship. It
has been a pleasure to work with you all and I would like to thank all of my laboratory
colleagues and friends, past and present for making it so: Kelly Heys, Holly Butler,
Lara Heppenstall, Liz Hill, Adil Ahmadzai, Junyi Li, Georgios Theophilou, Blessing
Obinaju, Alana Mitchell, Simon Fogarty, Valon Llabjani and Julio Trevisan. Thanks
also to Debra Hurst for providing technical support.
I am also grateful to the farmers and Rangers at Crake Trees, Whinton Hill and
Pennington Flash for allowing me to collect samples from their ponds and taking an
interest in the research I was carrying out.
Finally, I would like to thank my husband Jim for his endless support and patience,
particularly when times have been tough, and to my family and friends outside of
Lancaster for their encouragement, cups of tea and biscuits.

Declaration
I declare that this thesis is my work and has not been submitted for the award of a
higher degree or qualification at this university or elsewhere.

III
Contents

Title page I

Abstract II

Acknowledgements III

Declaration III

Contents IV

List of tables and VI
figures

List of VII
abbreviations

Chapter 1. General Introduction 1

Chapter 2. Biospectroscopy as a tool to monitor subtle effects of
environmental stress on the early life stages of the
64
Common frog: a pilot study.

Rebecca J. Strong, Crispin J. Halsall, Martin
Ferenčík, Kevin C. Jones, Richard F. Shore and Francis
L. Martin

Manuscript for submission

Chapter 3. Using biospectroscopy to monitor amphibian health: a
three year study.

107
Rebecca J. Strong, Crispin J. Halsall, Kevin C. Jones,
Richard F. Shore and Francis L. Martin

Manuscript for submission
IV
Chapter 4. Biospectroscopy reveals the effect of varying water
quality on tadpole tissues of the common frog (Rana
154
temporaria).

Rebecca J. Strong, Crispin J. Halsall, Martin

Ferenčík, Kevin C. Jones, Richard F. Shore and Francis
L. Martin

Environmental Pollution 213 (2016): 322-337

Infrared spectroscopy detects changes in an amphibian
Chapter 5. cell line induced by fungicides: comparison of single 205

and mixture effects.

Rebecca J. Strong, Crispin J. Halsall, Kevin C. Jones,

Richard F. Shore and Francis L. Martin

Aquatic Toxicology 178 (2016): 8-18

Chapter 6. Discussion 236

Bibliography 245

Appendix List of publications from collaborative research
277

Conference abstracts

V
List of tables and figures
Table1. Examples of endpoints in amphibians commonly measured following
exposure to environmental contaminants.......................................................................5
Table 2. List of commonly used fungicides in the UK and their mechanism of
action.............................................................................................................................11
Figure 1. Simplified life cycle of the common frog, Rana temporaria......................14
Figure 2. Rana temporaria spawn (A) and tadpoles (B)............................................20
Figure 3. Rana temporaria photographed breeding....................................................21
Figure 4. Schematic of a Michelson interferometer.....................................................25
Figure 5. Representative raw FTIR spectra.................................................................26
Figure 6. Vibrational modes in infrared spectroscopy.................................................27
Figure 7. Representative spectrum of the fingerprint region with chemical peaks
tentatively assigned.......................................................................................................28
Figure 8. Sampling modes in FTIR spectroscopy........................................................30
Figure 9. Overview of commonly used pre-processing steps in IR spectroscopy.......32
Figure 10. Example outputs following PCA-LDA analysis.......................................37
Table 3. A list of commonly used methods for classification of datasets generated
from IR spectroscopy....................................................................................................38
Figure 11. Overview of the aims and objectives of the thesis.....................................42
Figure 12. Approximate locations of the study sites on a map of the UK...................43
Figure 13. A workflow of the procedure used to obtain ATR-FTIR spectra of tissue
samples of Rana temporaria tadpoles..........................................................................45
Figure 14. Flow diagram of conclusions, limitations and future research needs.......236
VI
List of abbreviations
Al: Aluminium
AMPA: Aminomethylphosphonic acid
ANN: Artificial neural networks
ANOVA: Analysis of variance
ATR: Attenuated total reflectance
BCI: Body condition index
Ca: Calcium
Cl: Chloride
CYP: Cytochrome P-450
CYP51: Sterol-14α-demethylase
DCM: Dichloromethane
DMSO: Dimethyl sulfoxide
DNA: Deoxyribonucleic acid
ESI+: Electrospray positive
FBS: Fetal bovine serum
Fe: Iron
FFS: Forward feature selection
FPA: Focal plane array
FSH: Feature selection histogram
FTIR: Fourier-transform infrared
GC-MS: Gas chromatography–mass spectrometry
HNO : Nitric acid
3
HSI: Hepatosomatic index
HW: Head width
ICP-OES: Inductively coupled plasma optical emission spectrometry
VII
IR: Infrared
IRE: Internal reflection element
K: Potassium
kNN: k-nearest neighbours
LC-MS: Liquid chromatography–mass spectrometry
LDA: Linear discriminant analysis
LDC: Linear discriminant classifier
LoQ: Limits of quantification
Low-E: Low-emissivity
MeOH: Methanol
Mg: Magnesium
MRM: Multiple reaction monitoring
MS-222: Tricaine methanesulfonate
MSMA: Monosodium methyl arsenate
Na: Sodium
NO -N: Nitrate
3
OP: Organophosphorus
OWC: Organic wastewater contaminant
PAH: Polycyclic aromatic hydrocarbon
PBDE: Polybrominated diphenyl ether
PC: Principal component
PCA: Principal component analysis
PCB: Polychlorinated biphenyl
PLS: Partial least squares
PLS-DA: Partial least squares-discriminant analysis
QCL: Quantum cascade lasers
VIII
QDA: Quadratic discriminant analysis
QoI: Quinone outside inhibitor
RNA: Ribonucleic acid
SG: Savitzky-Golay
SHE: Syrian Hamster Embryo
SIMCA: Soft independent modelling of class analogies
SO -S: Sulphate
4
SNR: Signal to noise ratio
SVL: Snout to vent length
SVM: Support vector machines
TBP: Tributylphosphate
TCEP: Tris(2-chloroethyl)phosphate
TCPP: Tris(1-chloro-2-propyl)phosphate
TEP: Triethylphosphate
TON: Total organic nitrogen
UPLC: Ultra performance liquid chromatograph
UV: Ultraviolet
IX
Chapter 1. General Introduction

Contents
1. Introduction ................................................................................................................ 3
2. Threats to amphibian health from pollution ............................................................... 4
2.1 Pollution from urban environments ...................................................................... 8
2.2. Pollution from agriculture ................................................................................... 9
2.2.1. Fungicides ................................................................................................... 10
3. Life stages of anuran amphibians and sensitivity to environmental pollutants ....... 13
3.1 Life cycle of anuran amphibians ........................................................................ 13
3.2. Embryos ............................................................................................................ 14
3.3. Tadpoles ............................................................................................................ 15
3.4. Amphibian cell lines .......................................................................................... 17
4. Amphibians as biological indicators ........................................................................ 18
4.1. Suitability of the common frog as a biological indicator .................................. 19
4.1.1. Identification ............................................................................................... 19
4.1.2. Reproduction .............................................................................................. 20
4.1.3. Routes of exposure to environmental pollutants ........................................ 21
4.1.4. UK research on the effect of pollutants on native amphibians ................... 22
5. Biospectroscopy ....................................................................................................... 23
5.1. Background to Fourier-transform infrared spectroscopy .................................. 24
1