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Influence of Trace-Metals on the Colour and Photostability
of Wool
by
Alison Lee King B. Sc
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
November, 2011
ACKNOWLEDGMENTS
I would express my sincere thanks to Dr. Keith Millington (CSIRO, Materials
Science and Engineering) who arranged for me to undertake this study and
provided support and encouragement throughout the process and also to Dr.
Takuya Tsuzuki (Deakin University) who has provided me help and important
advice relating to my candidature.
Grateful thanks are due to Dr. Alec Zwart (CSIRO Mathematics Informatics
and Statistics) for his advice and thorough explanation of statistical analyses, to
Dr. George Maurdev for answering my questions and patiently listening to my
ideas and to the expert in wool science, Dr. John Rippon, for reading through
my manuscript.
This study would not have been possible without the financial support of the
Co-operative Research Centre for Sheep Industry Innovation. The Sheep CRC
also provided training and the opportunity to learn new skills and meet some
inspiring people for which I am grateful. Many thanks to the team of analysts
at CSIRO Process Science and Engineering (ex CSIRO, Minerals), in particular
Cheryl McHugh and Sally Taylor, for competently analysing the trace metal
content of the samples and for their help and advice when needed.
I am grateful to my employer CSIRO, Materials Science and Engineering for
releasing me from my former project to undertake this research. I which to
acknowledge the contribution of the large team of scientists and technicians
who maintain the Sheep CRC’s Information Nucleus Flock and the team at the
Australian Wool Testing Authority who measured the fibre properties of the
wool samples. I would also like to acknowledge my previous supervisors at
CSIRO for teaching me the art and processes of scientific enquiry and all the
researchers in the field that have contributed to the body of knowledge that I
have been able to draw upon.
Finally, I would sincerely like to thank my network of wonderful friends and
family who continually offered me emotional support. In particular, my
treasured children Rohan and Stefanie, my mother, sister and Ern, who
encouraged me from the beginning and accepted seeing less of me, my dear
Jim who patiently listened and reviewed draft documents and my friends
Michelina, Kerryn, Pauline, Debbie and Farhana, for being there for me.
Although I cannot tell them personally, thank you to my Dad, whose enquiring
mind has now been stolen by Alzheimer’s disease, for instilling in me a love of
science and to my dear late husband Russell for always encouraging and
supporting me.
PUBLICATIONS ARISING FROM THIS THESIS
A. L. King and K. R. Millington
Measurement of light penetration through a simulated Merino fleece.
Animal Production Science, 2010. 50: p. 585-588.
K.R Millington and A.L. King
Measuring colour and photostability of small fleece wool samples.
Animal Production Science, 2010. 50: p. 589-592.
Fleet, K.R. Millington and A.L. King
Sunlight exposure caused yellowing and increased mineral content of wool.
Animal Production Science, 2010. 50: p. 300-308.
K.R. Millington, A.L. King, S. Hatcher and C. Drum
Whiter wool from fleece to fabric.
Coloration Technology, 2011. 127: p. 297-303.
FOR RUSSELL JOHN KING (1954 - 2006)
“I wish life was not so short,” he thought. “Languages take such a time, and so do all the things one wants to know about."
J. R. R. Tolkien
TABLE OF CONTENTS ABSTRACT.......................................................................................................i
CHAPTER 1. INTRODUCTION
1.1. INTRODUCTION ............................................................................... 1
1.2. FACTORS AFFECTING WOOL COLOUR ...................................... 2
1.3. PHOTO-OXIDATION OF WOOL ..................................................... 4 1.3.1. Photoyellowing ............................................................................. 4 1.3.2. Fibre morphology and photoyellowing ........................................ 6
1.3.3. Measurement of photoyellowing .................................................. 7 1.3.4. Weathering ................................................................................... 9 1.3.5. Assessing the extent of weathering .............................................. 9
1.4. METHODS TO PRODUCE WHITER FLEECE WOOL ................. 10
1.4.1. Genetic selection ........................................................................ 10 1.4.2. Protection of fleeces ................................................................... 12
1.5. METAL-CATALYSED OXIDATION OF PROTEINS ................... 13
1.6. METAL-CATALYSED OXIDATION OF WOOL .......................... 14
1.7. METAL-CATALYSED OXIDATION OF LIPIDS.......................... 16
1.8. TRACE METALS IN WOOL AND HAIR ....................................... 18 1.8.1. Trace metals from soil to wool ................................................... 18
1.8.2. Uptake and binding of metals to proteins in wool ...................... 20
1.8.3. Distribution of metals in wool .................................................... 21
1.8.4. Measurement of trace metal content in wool ............................. 22
1.9. SUMMARY ....................................................................................... 23
CHAPTER 2. DEVELOPMENT OF A PROTOCOL TO PREPARE
WOOL SAMPLES FOR TRACE METAL ANALYSIS
2.1. INTRODUCTION ............................................................................. 26
2.2. MATERIALS AND METHODS....................................................... 27 2.2.1. Plastic Laboratory Ware ............................................................. 27
2.2.2. Preparation of a control wool sample ......................................... 28
2.2.3. Cleaning and drying ................................................................... 29
2.2.4. Doping wool fibre....................................................................... 29 2.2.5. Digesting wool............................................................................ 30 2.2.6. Trace metal analysis ................................................................... 31
2.3. RESULTS AND DISCUSSION ........................................................ 31 2.3.1. Comparison between cleaning methods ..................................... 31 2.3.2. Quality control procedures ......................................................... 33
2.4. SUMMARY ....................................................................................... 36
CHAPTER 3. OXIDATION AND YELLOWING IN PHOTO-
IRRADIATED METAL-DOPED WOOL
3.1. INTRODUCTION ............................................................................. 37
3.2. MATERIALS AND METHODS....................................................... 39 3.2.1. Preparation of fabrics ................................................................. 39
3.2.1.1. Metal-doped wool ............................................................... 39 3.2.1.2. Lipid-depleted wool ............................................................ 39 3.2.1.3. Adjustment of fabric pH ...................................................... 40
3.2.2. Trace metal content of fabrics .................................................... 40 3.2.3. Determination of hydroxyl radical production in wool .............. 41 3.2.4. Irradiation of fabric to assess photoyellowing............................ 42 3.2.5. Determination of carbonyl groups in wool ................................. 43 3.2.6. Colour measurement ................................................................... 44
3.2.7. Assessment of tensile strength.................................................... 44
3.3. RESULTS AND DISCUSSION ........................................................ 45
3.3.1. Hydroxyl radical production in metal-doped wool .................... 45 3.3.2. Chelating agents and antioxidants .............................................. 46 3.3.3. Colour change after exposure to UVB ....................................... 49 3.3.4. The effect of pH.......................................................................... 52
3.3.5. Carbonyl production in photo-irradiated wool ........................... 54 3.3.6. Lipid-depleted wool.................................................................... 55
3.3.7. Fibre strength of photo-irradiated wool ...................................... 57 3.3.8. Possible sources of metal contamination of processed wool ...... 57
3.4. SUMMARY ....................................................................................... 59
CHAPTER 4. TRACE METAL CONTENT AND COLOUR OF
FLEECE WOOL
4.1. INTRODUCTION ............................................................................. 61
4.2. MATERIALS AND METHODS....................................................... 63 4.2.1. Selection, preparation and analysis of samples .......................... 63
4.2.2. Statistical analysis ...................................................................... 66
4.3. RESULTS AND DISCUSSION ........................................................ 66 4.3.1. Distribution and range of trace metal content ............................ 66 4.3.2. The relationship between trace metal content and wool colour . 69 4.3.3. Effect of age on trace metal content and wool yellowness ........ 73
4.3.4. Calcium, magnesium and wool colour ....................................... 74
4.3.5. Limitations of this study ............................................................. 79 4.3.5.1. Effect of environment ......................................................... 79 4.3.5.2. Analysing different regions along the wool staple .............. 79
4.4. SUMMARY ....................................................................................... 81
CHAPTER 5. A METHOD TO MEASURE THE COLOUR AND
PHOTOSTABILITY OF SMALL FLEECE WOOL SAMPLES
5.1. INTRODUCTION ............................................................................. 83
5.2. MATERIALS AND METHODS....................................................... 84 5.2.1. Preparation of fleece wool samples ............................................ 84 5.2.2. Colour measurement ................................................................... 86 5.2.3. Photostability of dry fibre ........................................................... 87 5.2.4. Photostability of wet fibre .......................................................... 89
5.2.5. Hydrogen peroxide assay ........................................................... 91 5.2.6. Statistical analysis ...................................................................... 92
5.3. RESULTS AND DISCUSSION ........................................................ 93 5.3.1. Comparison to the standard IWTO-56-03 method ..................... 93
5.3.2. Access to oxygen ........................................................................ 95 5.3.3. Repeatability of the photostability test for wet wool .................. 95
5.4. SUMMARY ....................................................................................... 96
CHAPTER 6. TRACE METAL CONTENT AND THE
PHOTOSTABILITY OF FLEECE WOOL
6.1. INTRODUCTION ............................................................................. 97
6.2. MATERIALS AND METHODS....................................................... 98
6.2.1. Selection of samples ................................................................... 98 6.2.1.1. Evaluating the photostability of dry wool ........................... 98 6.2.1.2. Evaluating the photostability of wet wool .......................... 98
6.3. RESULTS AND DISCUSSION ........................................................ 99
6.3.1. The effect of iron and copper content on photoyellowing ......... 99 6.3.1.1. Photoyellowing of dry wool ................................................ 99 6.3.1.2. Photoyellowing of wet wool ............................................... 99
6.3.2. The effect of initial yellowness on photoyellowing ................. 103 6.3.3. Calcium, magnesium, manganese and zinc content and
photostability .......................................................................................... 104
6.4. SUMMARY ..................................................................................... 106
CHAPTER 7. MEASUREMENT OF LIGHT PENETRATION
THROUGH A SIMULATED MERINO FLEECE
7.1. INTRODUCTION ........................................................................... 107
7.2. MATERIALS AND METHODS..................................................... 108 7.2.1. Preparation of the simulated fleece .......................................... 108 7.2.2. Measuring the depth of light penetration.................................. 108 7.2.3. Colour measurement and trace metal analysis ......................... 109 7.2.4. Staining with methylene blue to visualise damage................... 110
7.3. RESULTS AND DISCUSSION ...................................................... 110 7.3.1. The extent of light penetration through a fleece ....................... 110 7.3.2. The extent of damage along the fibre ....................................... 111 7.3.3. Trace metal content along wool fibre ....................................... 113
7.4. SUMMARY ..................................................................................... 114
CHAPTER 8. CONCLUSION ................................................................ 115
REFERENCES… ......................................................................................... 123
APPENDIX 1 Photostability of wool after exposure to UVA for 18 hours
while wet and the trace metal content (mg/kg) .............................................. 147
i
ABSTRACT
While wool has many valued qualities that are superior to other textile fibres,
its natural cream colour and tendency to yellow mean that wool cannot
compete with cotton and synthetics in the lightweight, trans-seasonal apparel
market for which bright colours and pastel shades are essential. Previous
studies to evaluate the mechanisms of yellowing identified that trace metals
within the wool fibre are associated with the production of free radicals and
yellowing in photo-irradiated wool. In biological systems, protein-bound
metals that are capable of single electron transfer can catalyse the production of
hydroxyl radicals that oxidise proteins and produce carbonyl species. This
present study quantified the production of hydroxyl radicals and carbonyl
species as well as the extent of yellowing in photo-irradiated metal-doped wool
fabric. Although the presence of metals did not influence carbonyl content, iron
increased hydroxyl radical production and both copper and iron increased the
rate of photoyellowing. Two methods were developed to determine if the
intrinsic levels of copper and iron had the same effect on the colour and
photostability of fleece wools: a protocol for the quantitative determination of
trace metal content in wool and a method to estimate the rate of wool
yellowing during exposure to an artificial light source. Iron and copper content
did not positively correlate with either wool colour or photostability. Instead, a
strong positive correlation of yellowness with the calcium and magnesium
content was observed in yellow wools, which is consistent with biofilm
formation by Pseudomonas bacteria that have previously been associated with
non-scourable staining of wool. In addition, photostability was negatively
correlated to zinc content.
The major portion of a full fleece is somewhat protected while the tips of the
fibre receive the maximum dose of sunlight. The tips also have higher levels of
iron which are likely to accelerate the rate of photoyellowing and photo-
tendering.
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CHAPTER 1. INTRODUCTION
1.1. INTRODUCTION
Sheep were domesticated over 10,000 years ago and initially their wool was
coarse and pigmented with melanin. Selective breeding of white, non-
pigmented sheep occurred after the development of dyeing in the Middle East
at around 1000BC [1]. The result of genetic selection to enhance comfort and
visual appeal has produced wools that today, are softer and whiter. Wool has
now become an important Australian commodity and is traditionally sold to
northern hemisphere markets for the manufacture of winter-weight outerwear
which is usually dyed with darker colours.
As fashion trends evolved, the desire for brighter, more vibrantly coloured
clothing has developed, especially in the next-to-skin trans-seasonal knitwear
market. Fabrics must be as white as possible to achieve brilliant shades and
wool’s natural cream colour means that it compares unfavourably with cotton
and synthetics. While bleaching can increase the reflectance, and hence the
whiteness of wool, bleached wool is still yellower than other apparel fibres;
cotton, nylon and polyester (Figure 1.1).
Like silk, wool is predominantly comprised of protein and is damaged and
yellowed by sunlight. Wool’s colour and poor photostability, compared to
cotton and synthetics, has prompted an extensive body of research to identify
the factors which influence colour, to elucidate the mechanisms of
photoyellowing and to develop methods to improve the colour and
photostability of wool [2, 3]. These previous studies identified that trace metals
present in the wool fibres are involved in mechanisms that are initiated by
exposure to light [4-6]. This present enquiry further examined the influence of
trace metals on the colour and photostability of wool.
CHAPTER 1
2
Figure 1.1 Comparison of the reflectance spectra of undyed apparel fabrics [1].
1.2. FACTORS AFFECTING WOOL COLOUR
Previous studies have shown that the colour of scoured wool from Australian
Merino sheep is moderately heritable [7-9]. Genetic correlation with wool
colour is most likely from inherited variation in natural pigments within wool
fibres, excretions from the skin [10] and fibre diameter, which is positively
correlated to yellowness [8, 11, 12]. Genetic responses to environmental
influences can affect the amount of suint produced. Genetic responses also
affect the ratio between the amount of wax (secreted by the sebaceous glands)
and suint (secreted by the sudoriferous glands), both of which can influence
wool’s colour and its propensity to yellow [13].
Nutritional differences can alter the colour and amino acid composition of
Cashmere goat fibre [14] and the addition of tannins to the diet of sheep
changes the amino acid composition and reduces wool yellowness [15]. In
contrast, cysteine supplementation [16] and variation of the source and amount
of dietary protein for sheep does not influence wool colour [17]. Changes in the
level and source of protein in the diet alters suint composition without
influencing fleece discoloration [17]. However the protective effect of suint on
30
40
50
60
70
80
90
380 420 460 500 540 580 620 660 700 740
Rele
cta
nc
e (
%)
Wavelength (nm)
natural wool
peroxide bleached wool
cotton
bleached cotton
nylon
polyethylene terephthalate (PET)
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wool from exposure to light [18] may vary as a consequence of differences in
suint composition.
The bacterial pathogen Pseudomonas aeruginosa, which is part of the
ubiquitous microbial population present in the fleece, is associated with fleece
rot [19] which is a mild dermatitis that is induced by warm temperatures and
high levels of moisture [20]. Pseudomonas aeruginosa produce highly
coloured phenazines that stain wool and cannot be removed by scouring [21].
The development of the unscourable coloration, commonly called “canary
yellowing” occurs both on the sheep and during storage of the wool [13].
Environmental influences strongly affect the colour of wool. Pigments in
plant material and excreta, contact with reagents to treat external parasites and
exposure to heat and alkali either discolour the fleece or induce wool yellowing
[22]. Sunlight is an important environmental influence on the colour of fleece
wool since it causes oxidation, resulting in yellowing and tendering of the
fibre. The exposed, oxidised tips of the wool fibre become brittle and can break
off during processing [23] resulting in a loss of productivity. If they are not
removed and are incorporated in yarn or fabric, the damaged wool takes up dye
at a different rate to the undamaged fibre resulting in uneven, tippy dyeing
[24]. Photo-oxidised wool is therefore a poor substrate for the manufacture of
wool products because of the loss of tensile strength, uneven dyeing properties
and the inability to achieve photo-stable, bright and pastel colours.
The trace metal status of sheep has some influence on wool colour. Fleeces
which are more susceptible to yellowing have slightly lower levels of zinc and
higher levels of copper [13] and dipping sheep with zinc has been reported to
reduce yellowing [25]. Hypocuprosis in sheep causes “steely wool” which, in
unpigmented wool, is more lustrous and whiter than normal wool.
Depigmentation of wool from coloured copper-deficient sheep is a result of
impaired copper-dependant synthesis of dihydroxyphenylalanine (DOPA) from
tyrosine, a precursor for the pigment melanin. Hypocuprosis is easily treated
with supplements when deficiencies are identified, and nowadays severe
deficiencies of copper in sheep are rare. However, areas of copper-deficient
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4
soils have been identified in South Australia and Merino sheep in these regions
produce whiter wool compared to sheep grazing on pastures with higher copper
content [11]. There are no reported studies of a relationship between the colour
of non-pigmented wool and its trace metal content.
1.3. PHOTO-OXIDATION OF WOOL
1.3.1. Photoyellowing
Exposure of wool to solar radiation induces both yellowing and bleaching
concurrently, although yellowing is the long-term dominant effect. Photo-
bleaching is initiated when wool is exposed to radiation at wavelengths
between 380 and 475 nm due to the destruction of pre-existing yellow
chromophores present in the fibre. Photoyellowing occurs as a result of the
production of yellow chromophores at wavelengths less than 320 nm [26] and
the rate of yellowing is greater in wet wool than in dry wool [2].
The mechanisms of photoyellowing are complex and are not fully elucidated,
however a large number of studies have provided insight into the possible
mechanisms [2, 3]. Photoyellowing is initiated when amino acids in wool
proteins absorb UV radiation. The aromatic amino acids all absorb UV,
however tyrosine, tryptophan and phenylalanine absorb the greatest amount of
energy between 250 and 300 nm [27, 28], the extent of which depends on their
position in the protein. Amino acids that absorb UV are either completely or
partially destroyed [29].
In an early theory, photoyellowing of wool after exposure to UV was attributed
to the decomposition of cystine [30]. However, the correlation between the rate
of yellowing and the oxidation of cystine is poor [31, 32] and there is little
difference in the rate of photoyellowing between cystine-depleted wool and an
untreated wool control [33]. Also, silk photoyellows to the same extent as wool
despite containing very little cystine [34], (26 mol/g) compared to wool (1048
mol/g) [35]. While cleavage of the disulfide bonds between cystine residues
is responsible for the loss of tensile strength of wool during weathering and
CHAPTER 1
5
exposure to sunlight [36], coloured oxidation products of cystine are not major
contributors to photoyellowing.
The strong correlation between the rate of degradation of tryptophan residues
with wool yellowing after irradiation with UV light supports the theory that
tryptophan is important in for photoyellowing [37]. Tryptophan degrades after
exposure to light [38] and enzymatic digestion of photo-irradiated wool
enriched with radio-labelled tryptophan yielded yellow products from the
tryptophyl residues [39]. When exposed to UV radiation phenylalanine
residues are hydroxylated to tyrosine [40] and energy absorbed by tyrosine is
transferred to tryptophan via triplet-triplet transfer [28]. Triplet tryptophan and
tyrosine species transfer electrons to an acceptor to form tryptophan and
tyrosine free radicals [41]. Triplet tryptophan also reacts with ground state
oxygen to produce reactive oxygen species that can react with proteins to form
yellow oxidation products [27]. However treatments that deplete unbleached
wool of tryptophan do not affect the rate of photoyellowing [42]. This indicates
that other significant processes contribute when tryptophan is not present.
Reactive oxygen species, such as singlet oxygen [43] and hydroxyl radicals,
[44] have been detected in photo-irradiated wool. It is unlikely that singlet
oxygen is involved in photoyellowing because wet wool yellows far more
rapidly than dry wool, and the lifetime of singlet oxygen in water is very short
[3]. Also, the lifetime of singlet oxygen increases in deuterated water but the
rate of photoyellowing does not change [3]. Hydroxyl radicals are highly
reactive species which readily react with amino acid residues and are likely to
be involved in the production of yellow degradation products in photo-
irradiated wool [44].
Yellow chromophores that have been characterised in photo-irradiated wool
include -carboline carboxylic acids [45] and oxidation products of tryptophan
and tyrosine; hydroxytryptophan, dityrosine and dihydroxytyrosine,
dihydroxyphenylalanine and kynurenines [46, 47]. Kynurenine can form cross
links with histidine, and to a lesser extent lysine and tryptophan, to form
coloured compounds in the crystallin proteins of eye lenses [40, 48] and this
CHAPTER 1
6
mechanism may also occur in photo-oxidised wool. Photoyellowing has also
been attributed to the formation of conjugated products [3], possibly via the
tryptophan residues in keratin proteins [49].
Carbonyl species in the form of keto acids are produced during photo-oxidation
of the amino acid residues in wool, however the level of carbonyl species in
wool is not correlated to the rate of photoyellowing [50]. Although the
carbonyl content is not related to the extent of photoyellowing, keto acids
may undergo facile aldol condensation [51] and are therefore possible
intermediates in the production of yellow polymeric chromophores [50].
Carbonyls can also react with tryptophan to produce coloured carbolines [51]
and these species have been identified in the weathered tips of wool [45].
1.3.2. Fibre morphology and photoyellowing
The three major morphological components of wool fibre are the cortex, the
cuticle and the cell membrane complex [52] (Figure 1.2). The external cuticle
is a single layer of overlapping cells containing highly cross-linked, cystine
rich proteins and the surface is covered by the epicuticle. The inner cortex,
constitutes 90% of the mass of the fibre and is comprised of ortho and para
cortical cells. The cell membrane complex is a matrix of proteins and lipids
between the cortical cells that act as a glue to bind the internal cells [52] .
Cuticle material can be mechanically separated from the cortex. Two separate
studies have shown conflicting results, and that the extent of yellowing after
exposure to UVB is greater in the cuticle [53] and the cortex [47]. The
conclusion of the latter study is more probable given that cortical cells have a
higher level of aromatic amino acid residues than the remainder of the fibre
[47], that tyrosine is only found in the cortex [54] and that the extent of
photoyellowing of isolated ortho cortical cells is the same as that of whole
wool fibre [30]. The penetration of UVA (320-400 nm) and visible light
extends to the cortex of the wool fibre whereas the penetration of UV at
wavelengths less than 320 nm is restricted to the cuticle [2]. Since the intensity
of UVB (280-320 nm) in terrestrial solar radiation is approximately 6% of the
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7
intensity of UVA, and that there is a greater mass of proteins in the cortex,
photoyellowing of the cortex is likely to be of greater significance in fleece
wools.
Figure 1.2 Structure of a wool fibre (CSIRO Materials Science and Engineering).
1.3.3. Measurement of photoyellowing
Although wool yellowness can be assessed by visual comparison to a set of
standards, it is more accurately assessed using a reflectance spectrophotometer
with a standard illuminant. The CIE tristimulus values X (red), Y (green),
which indicates brightness, and Z (blue) [55], are calculated from the
percentage reflectance values for each sample at each wavelength. These three
values describe the colour of substrate under a specified illuminant and are
related to the response of the receptors in the human eye [56]. Higher values
indicate greater brightness and hue. The degree of yellowness can be expressed
by several indices and the most commonly used value for wool is the
difference between the Y and Z tristimulus values (Y-Z).
Wool fibres are constrained when prepared as yarns or fabric, and colour can
be measured reproducibly by placing a test specimen directly against the
observation port of a spectrophotometer. The colour of loose fibre cannot be
measured this way because fibres tend to protrude into the aperture causing
errors in the measurement and loose fibres can fall inside the instrument. There
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8
will also be variations in the sample density which significantly affects colour
measurements on scoured wool samples [57].
The standard test method for measuring the colour of scoured wool fibre is
IWTO-56-03 [58]. This method requires 5g of loose wool compressed to
constant density against a glass aperture. Using this method, typically the
whitest wools have yellowness values (Y-Z) of approximately six and average
wools have Y-Z values between 7 and 8. The colour of Merino wools with Y-Z
less than 8.5 is considered acceptable, whereas Y-Z values greater than 11
indicate heavily yellowed wools that are unsatisfactory [56].
Estimation of the extent of photoyellowing requires measuring the change in
colour before and after a set period of irradiation using a UV source at a
constant distance from the sample (∆ Y-Z). It is critical that fibres are
completely constrained during the entire process of measurement and
irradiation and that the fibres exposed to the radiation remain facing the
measured surface. The movement of fibres in fabrics and yarns wound onto a
suitable former is limited and therefore these constructions are very convenient
for measuring photoyellowing. For wool fabric, the change in yellowness after
irradiation is linearly related to the initial yellowness [59].
While suitable for colour measurement of loose fibre, the IWTO-56-03 method
is inappropriate for assessment of wool photostability because the compression
cell is integrated with the spectrometer and cannot be removed during
irradiation of the samples. A method for preparing loose wool fibre for
assessment of photoyellowing entails securing the fibres on a card and
covering the fibres with a material that is transparent to UV and visible light.
The yellowness after irradiation is a function of the initial yellowness and
exposure time and the relationship can be expressed as Y final = a + b Y initial + c
log t [37]. However, uneven densities of fibre on the card and fibre movement
during measurement and irradiation greatly reduce the accuracy of this method
compared to measurements on fabric.
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9
A more reliable method is to pack loose wool fibre into individual compression
cells, because test specimens can always be packed to the same density and
fibre movement during testing is completely restricted. This method has been
used to compare the rate of photobleaching of wool fibre in sunlight and with
an artificial light source [60]. In this process, the cells were modified
polycarbonate containers manufactured by replacing the base of the container
with 2 mm thick picture glass. In this present study, a new method suitable for
measuring the photostability of small samples of both wet and dry loose wool
has been developed which uses commercially available, inexpensive and
disposable polymethyl methacrylate cuvettes as compression cells.
Development of this method is described in Chapter 5.
1.3.4. Weathering
Degradation of wool due to exposure to sunlight, together with the effect of
wind and rain, induces colour changes and loss of tensile strength. This is due
to the oxidation and hydrolysis of the proteins, and an increase in the
brittleness of the fibre due to protein cross linking [61]. The disulfide linkages
that crosslink the protein structure of the wool fibre are cleaved and cystine is
oxidised to cysteic acid. Amide and carbonyl groups are produced by oxidation
and bond cleavage of proteins and the photo-oxidation of alanine, glycine,
proline, glutamic acid, tyrosine, [50] and serine, threonine and cystine residues
[62]. The oxidation products N-formylkynurenine [63] and carbolines [45]
are photoreactive and can further sensitise the degradation of wool. The levels
of histidine and tryptophan are also significantly reduced [31], indicating that
the wool structure is extensively damaged due to exposure to the environment
[64].
1.3.5. Assessing the extent of weathering
Accelerated tests have been developed to assess the extent of damage caused
by weathering. Test specimens are exposed to a standard light source and some
methods also include humidity control. The extent of the loss of tensile
strength of wool fabric from artificial weathering can be quantified by
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10
measuring the changes in breaking or burst strength [65] and other mechanical
properties such as shear stiffness, bending rigidity, compressional linearity and
surface friction [64]. Physical damage caused by weathering can also be
assessed by microscopic examination [66, 67].
Cleavage of disulfide crosslinks in weathered and photodamaged wool
increases the degree of swelling of wool fibre in aqueous buffers [67] and
increases the solubility in urea/bisulphite or alkali solutions [68]. These
properties can be used as an index for the extent of chemical damage.
Photochemical modification of the outer surface of wool fibre can be visualised
using Kiton Fast Red G (C.I. Acid Red 1) [67], Erional NW (C.I. Food Red 17)
[24] or Methylene Blue G (C.I. Basic Blue 9) staining tests [67, 69]. These
three dyes have a greater affinity for oxidised wool and the tips of fleece wool,
which have the maximum exposure to environment, are stained to a greater
extent than the root and mid-section of the fibre [54].
1.4. METHODS TO PRODUCE WHITER FLEECE WOOL
1.4.1. Genetic selection
Heritability is an estimate of how much differences in observed traits are due to
genetic variation. Values less than 0.1 indicate low heritability, and values
approach 1.0 when genetic effects are maximised. Early estimates indicate that
the colour of both greasy (0.53 ± 0.01) and scoured (0.45± 0.08) [8] Australian
Merino wool is heritable and that selection for these traits would not have any
adverse correlations with other desirable traits [7]. A later study has shown
lower estimates for clean colour (yearlings 0.25, adults 0.29, ± 0.02-0.04) and
greasy colour (yearlings 0.33, adults 0.40, ± 0.02-0.04) [9], while a more recent
study estimates that the heritability of clean wool colour is as high as 0.70 ±
0.11 [70]. Despite the variation in values, positive estimates indicate that
genetic selection for scoured colour would expedite an improvement to the
average whiteness of the wool clip.
CHAPTER 1
11
The Co-operative Research Centre for Sheep Industry Innovation manages an
experimental flock of 5000 ewes, approximately 1500 of which are Merinos.
The sheep consist of range of breeds and bloodlines and are located over eight
research sites and four states (Table 1.1) (Figure 1.3). The Sheep CRC
Information Nucleus Flock (IN Flock) will be maintained for five years and
data will be collected and evaluated to expand on the knowledge of genetic
influences on wool and meat (carcass) traits. As well as genetic influences, it is
anticipated that data from the IN Flock will provide further insights into the
effect of environmental and geographical influences on wool colour.
IN Flock location Site
number
Merinos in the IN Flock
2007 2008 2009
Kirby Research Station, University of New
England, Armidale, NSW IN 01 296 480 397
Trangie Agricultural Research Centre,
NSW DPI, Trangie, NSW IN 02 - 209 326
Cowra Agricultural Research & Advisory
Station, NSW DPI, Cowra, NSW IN 03 148 230 258
DPI Rutherglen Centre, DPI Vic,
Rutherglen, Vic IN 04 122 272 298
DPI Hamilton Centre, DPI Vic, Hamilton,
Vic IN 05 122 214 245
Struan Research Station, SARDI, Struan,
SA IN 06 155 189 169
Turretfield Research Station, SARDI,
Rosedale, SA IN 07 182 341 305
Great Southern Agricultural Research
Institute, DAFWA, Katanning, WA IN 08 263 617 474
Total 1288 2552 2472
Table 1.1 Merinos in the Sheep CRC Information Nucleus Flock.
CHAPTER 1
12
Figure 1.3 Locations of Information Nucleus (IN) flocks in Australia (CRC for
Sheep Industry Innovation).
1.4.2. Protection of fleeces
The extent of wool yellowing depends on which part of the sheep the wool is
grown. The scoured wool colour of back wool can have a yellowness of 0.9
units greater than wool grown on the flanks [71] due to differences in exposure
to the environment. In an effort to improve the colour of wool and reduce
damage and yellowing, methods have been developed to reduce the influence
of ultraviolet radiation. These include rugging sheep, which produces whiter
and brighter wool [72], and the application of UV absorbing treatments to
newly shorn sheep that achieves an efficiency of 70% of that attained by
rugging [71]. The ultimate protection from UV, dust and contamination from
vegetable matter is to rug sheep and house them in sheds. This method is
commonly used in New Zealand and is also used by the Australian Sharlea
Society to produce very clean, white, 12 to 15.5 micron wool from Saxon
Merino sheep.
A more comprehensive knowledge of the mechanisms of photo-oxidation of
wool provides the opportunity to develop processes and treatments to minimise
or prevent it. Also, a better understanding of the effect environmental
influences and the nutritional and trace metal status of sheep have on wool
colour is required to optimise the production of whiter wool from genetically
selected sheep.
CHAPTER 1
13
1.5. METAL-CATALYSED OXIDATION OF PROTEINS
Oxidative modification of proteins in vivo is caused by the attack on proteins
by reactive oxygen species and results in functional changes such as
fragmentation, proteolysis, protein-protein cross linking and conformational
changes and aggregation. The most significant mechanism of protein oxidation
in biological systems is catalysed by metal/protein complexes [73]. As well as
being constituents and activators of enzymes, metals which are capable of
single electron transfer in redox systems, particularly iron and copper, can
catalyse the production of hydroxyl radicals from oxygen or hydrogen peroxide
within cells. These hydroxyl radicals ultimately oxidise and damage proteins
[74]. Metal catalysed oxidation of proteins is associated with natural ageing
and premature ageing (progeria) [75] and diseases involving the production of
amyloid plaque in Alzheimer’s disease [76], the oxidation of crystallin
proteins in human lenses [77] and the degeneration of neurotransmitters in
Parkinson’s Disease [78]. All amino acid residues are capable of oxidation by
hydroxyl radicals, although cystine and methionine [79] as well as proline,
arginine and lysine [80] are particularly sensitive.
Metal-catalysed oxidation is based on the Haber-Weiss reaction, whereby ferric
ions are reduced to ferrous ions by reaction with superoxide (1) and Fenton’s
reaction where iron reacts with hydrogen peroxide to form hydroxyl (2) and
peroxy (3) radicals.
Net reaction:
Fe+3 + O2¯ Fe+2 + O2 (1)
Fe+2 + H2O2 Fe+3 + OH + OH ¯ (2)
Fe+3 + H2O2 Fe+2 + OOH + H+ (3)
H2O2 + O2¯ OH + OH ¯ + O2
CHAPTER 1
14
The accepted mechanism in metal-catalysed oxidation in biological systems
requires an electron donor to reduce any trivalent metal ions from M3+
to M2+
.
Divalent metal ions bind to the amino metal-binding sites on the protein and
the metal/protein complex reacts with oxygen or hydrogen peroxide to produce
hydroxyl radicals which attack the amino acids in close proximity to the metal
binding site. The hydroxyl radical abstracts a proton from the carbon atom
adjacent to the amino group forming a carbon-centred radical. This radical
donates its unpaired electron to the iron/protein complex to reduce the metal
and form an imino group. The divalent metal ion is released and the imino
group hydrolyses to form a carbonyl species [81]. The extent of oxidation can
be monitored by quantifying the carbonyl content of proteins [80].
The carbonylated protein site is located on the surface of the protein and is a
Lewis base with an increased ability to bind metals. The enhanced affinity for
metals increases the likelihood of hydroxyl radical production thereby
increasing the probability that carbonylatable sites close to an existing carbonyl
species are oxidised. Carbonyl species are therefore found in clusters on the
protein [82].
1.6. METAL-CATALYSED OXIDATION OF WOOL
There is evidence that metals, particularly iron and copper which have redox
activity, are involved in the processes of photo-oxidation of wool. Metals bind
strongly to proteins. Iron and copper, which are bound to the proteins in wool,
increase the rate of photoyellowing [6]. Also, similar species in the mechanism
of metal-catalysed oxidation of proteins in biological systems are produced in
photo-irradiated wool.
Hydroxyl radicals are produced in metal-catalysed oxidation of proteins and
they are also produced in wool during irradiation with UVA [44]. It is likely
that metals are involved in the oxidation of wool proteins because hydroxyl
radical production decreases with the addition of an iron chelator [44]. Thiol
groups can act as electron donors which are required for metal-catalysed
CHAPTER 1
15
oxidation [83] and these species are present in oxidised wool from cleavage of
cystine residues. Iron (III)-sulphur complexes are involved in photo-initiated
free radical formation [4]. Copper (II) wool keratin complexes, in the presence
of hydrogen peroxide, also catalyse the production of the intermediate
superoxide radical which results in hydroxyl radical formation and ultimately
wool protein degradation [84].
Highly reactive hydroxyl radicals abstract protons from adjacent carbon atoms
in proteins and carbon centred radicals have been detected in photo-irradiated
wool [85]. Metal ions that are bound to carboxyl groups, photosensitise
decarboxylation of the terminal amino acids or the glutamic and aspartic
residues, and this reaction is important for the generation of carbon-centred
free radicals [86]. Abstraction of hydrogen atoms from photo-irradiated
peptides of alanine and glycine is facilitated by the presence of iron [87].
Copper and iron ions, that are complexed to wool, increase the production of
glycyl free radicals [5, 6], indicating the likelihood that metal catalysis is an
important mechanism in the generation of free radicals in wool.
In the presence of iron (II) and hydrogen peroxide, tryptophan is oxidised and
the tyrosine residues in human serum albumin and superoxide dismutase cross-
link to form dityrosine [88]. Dityrosine and kynurenines, which are oxidation
products of tryptophan, are also found in photoyellowed wool [40, 46]. In
addition, carbonyl species are products of metal catalysed oxidation of proteins
and these species have been detected as keto acids in photo-irradiated wool
[50, 89].
Conversion of the triplet states of tyrosine and tryptophan to free radicals
requires the transfer of an electron to a species capable of accepting electrons,
such as oxygen or disulfide groups [41]. Protein-bound metals, which are
capable of redox transfer of electrons, may also act as electron acceptors.
However, metal-catalysed oxidation of proteins requires an aqueous medium to
enable Fenton-type reactions to occur and it is likely that this mechanism only
occurs in wet irradiated wool, which yellows at a far greater rate than dry wool
[3].
CHAPTER 1
16
1.7. METAL-CATALYSED OXIDATION OF LIPIDS
The external lipids that coat wool fibre are comprised of approximately 10,000
different species of lipids, cholesterols and fatty acids [90]. Exposure to the
environment oxidises the lipids in wool wax and changes the composition of
the lipid matrix, particularly at the tip of the wool fibre which has a higher
proportion of fatty acids and oxidation products than the remainder of the fibre
[91].
Wool wax contains lipids of different polarity. The polar lipids include
cholesteryl sulphate, ceramides, hydroxyacids, diols and oxidation products of
cholesterol and the medium-polarity lipids are free fatty acids, fatty alcohols,
cholesterol and hydroxyl cholesterol esters. The low polarity lipids are
triacylglycerides and aliphatic and steroidal monoesters [92]. Changes in
composition after exposure make the external lipids more polar, making it
possible to almost completely remove them during aqueous scouring. The “f”
layer, a thin external layer of fatty acids that is covalently bonded to the
epicuticle is not removed by aqueous scour liquors [52]. The principal
covalently bound lipid that has been identified on wool fibre [93] and human
hair [94] is 18-methyl-eicosanoic acid.
The thickness of the external wax layer on unscoured natural wool fibres is
insufficient to provide a barrier for water vapour and oxygen and does not
provide protection to the wool fibre against exposure to sunlight and weather
[18]. Embedded dust from exposure to the environment can partially screen the
fibre from sunlight and weather but also provides a source of metal ions that
can be adsorbed by the lipids in the wool wax and proteins within the fibre.
Internal lipids surround the inner cellular structures within the fibre and are
approximately one percent of the total mass of wool fibre [52]. The internal
lipids have a similar composition as unoxidised wool wax and are comprised of
sterols, fatty acids, polar lipids and phospholipids. These lipids are not
removed by aqueous scour liquors and their extraction from wool requires
organic solvents.
CHAPTER 1
17
Lipids readily oxidise to form peroxides and the mechanism can be initiated by
reactive oxygen species or any other species capable of abstracting a proton
from a methylene carbon (Figure 1.4). Lipid peroxyl radicals can react with
and damage proteins and induce protein cross linking [95], fragmentation and
increase the production of protein carbonyls [96]. Lipid oxidation contributes
to oxidative damage in cells [81], spoilage of foodstuffs [97] and fracture of
wool fibres after exposure to light [66].
Figure 1.4 Mechanism of lipid peroxidation.
The rate of lipid oxidation increases after exposure to UV [98], particularly
UVA [99], and is sensitised by the combination of metal ions and reducing
agents [100, 101] such as thiolated species and chromophores such as
chlorophyll [102, 103] which can initiate the production of reactive oxygen
species.
Copper-catalysed lipid peroxidation is attributed to the deterioration of colour,
texture and flavour of foodstuffs. A copper impurity as little as 0.5 ppm in
brining liquors is responsible for lipid oxidation and the change in colour of
fish meat [104]. The natural level of copper in milk is sufficient to catalyse the
oxidation of milk lipids causing the flavour to alter [105]. Lipid peroxidation is
reduced with the addition of iron chelators [106] and protection from UV.
H
R
+
ROS
H2O R
OO
R
O2
+
OOH
R
H
R
lipid lipid
radical
lipid
peroxide
peroxyl
radical
initiation
propagation
H
R
+
ROS
H2O R
OO
R
O2
+
OOH
R
H
R
lipid lipid
radical
lipid
peroxide
peroxyl
radical
initiation
propagation
CHAPTER 1
18
It is feasible that UV, in the presence of iron and copper ions, may induce
peroxidation of the lipids in wool and that these mechanisms are initiated by
plant and protein contaminants that contain UV-absorbing chromophores.
However since the level of lipids in wool is low, the importance of lipid
peroxidation on photo-oxidation of proteins in wool may be of minor
importance.
1.8. TRACE METALS IN WOOL AND HAIR
1.8.1. Trace metals from soil to wool
Trace metals are essential for biochemical activity and are always found in
natural protein fibres. Human hair and wool fibres have similar chemical and
physical structure and are formed by the same biochemical process therefore
similar mechanisms of metal uptake and migration occur in both fibres.
Exogenous metals are either occluded on the surface of the wool fibre from
airborne dust [107] or bound to the proteins in wool by adsorption from soil
and water. Dust contamination is a major source of trace metals, particularly
for the exposed tips of the fibre which have a higher metal content than the
remainder of the fibre [108]. Adsorption of metals into the fibre increases when
the epicuticle is damaged [109].
Metals are ingested from fodder, soil and drinking water and the nutrition of
ruminants and their trace metal status is largely dependent on the composition
of the soil [110, 111] (Figure 1.5). However, the mobility of trace metals and
their bioavailability to plants depends on physical and chemical processes such
as the degree of compaction, pH and the ion exchange capacity of the soils.
Microbe populations within the soil can convert insoluble elements to soluble
products [112] and complex iron, zinc and copper, thereby increasing their
availability to plants.
CHAPTER 1
19
The uptake of metals by plants is influenced by the concentration and solubility
of trace metals in the soil, the morphology of the roots, the degree and structure
of root development, the ion exchange capacity and the moisture content of the
soil. The amount of trace metals in the diet of sheep will therefore depend on
the concentration in the herbage and this varies with climate, soil type, season,
plant species and the stage of plant growth. The bioavailability of trace metals
to sheep also depends on the digestibility of the forage and the preference of
sheep to different plant species. Soil is an additional source of trace metals
when consumed by sheep, either directly or attached to plant roots, and this can
be important for nutrition when plant sources are limited [113].
Figure 1.5 Sources of trace metals in sheep. Redrawn from [114].
Certain ingested trace metals in the circulating blood and cellular fluids are
ultimately incorporated into hair and wool as it grows in the follicle. These are
termed endogenous trace elements. The matrix cells and papilla at the base of
the follicle, together with surrounding blood and lymph and extracellular
fluids, provide the growing hair with nutrients and dissolved trace metals. The
sweat (suint) produced by the eccrine glands and oils (wax) produced by the
sebaceous glands also contain trace metals that can be adsorbed into the wool
fibre as it is emerging from the follicle [115]. Epithelial cells which are
CHAPTER 1
20
desquamated from the outer surface of the skin [116] and wound exudates
[117] are minor sources of trace metals in wool and hair.
Zinc is the major element required by sheep for keratinisation and wool growth
[118] and deficiencies cause wool to become thinner, lose crimp and shed
[114] and the production of wool wax and suint increases [118].
Approximately 20-30% of the total zinc present in a sheep is bound to a full
fleece [119]. The level of zinc in wool is relatively constant regardless of
dietary intake [118, 120] and dosing healthy sheep with zinc has little effect on
wool quality [120].
Copper is also required for growth and keratinisation of hair and wool, possibly
in ionic form or as the copper-containing enzyme sulfhydryl oxidase which
catalyses the oxidation of the thiol or cysteinyl residues to disulphide linkages
[121]. As well as affecting wool colour, hypocuprosis causes wool to grow
with low crimp and low tensile strength and have more cysteic acid and less
tryptophan than wool from sheep with normal copper status [68]. Copper and
sulphur in wool are positively correlated and higher copper levels are
associated with the production of finer wool [122] confirming that copper is
involved in the mechanism of wool growth.
There is little variation in zinc and copper content along the length of the wool
or hair fibre, suggesting that the levels of these elements are controlled
metabolically and that there is little contribution from environmental
contamination [123, 124].
1.8.2. Uptake and binding of metals to proteins in wool
The extent of adsorption of metals into wool from exogenous aqueous sources
depends on the time and temperature of exposure and the concentration and pH
of the metal salt solution. Adsorption is low under acidic conditions when
metal binding sites are protonated. More alkaline suint, which can range in pH
from approximately 6.7 to 9.7 [125], is likely to promote the uptake of metals
since adsorption increases with pH [126]. The uptake of metals in wool from
CHAPTER 1
21
aqueous solutions of metal salts is also dependant on the oxidation state of the
metal cation and possibly the associated anion [127].
Metals bind strongly to proteins and become relatively inert once they are
incorporated into the wool fibre. The order of binding affinity of metals to hair
proteins is Ca<Mg<Mn<Fe<Ni<Cu<Zn [128] and is likely to be similar to the
keratins in wool fibres. Copper (I) [129] and iron [130] preferentially bind
to the thiol groups of cysteine and can form stable mercaptides which are
incorporated into the hair shaft [116]. Copper (II) [131, 132] and zinc bind to
carboxyl groups, amines, thiolates, ionised peptides and carboxylates [129] and
copper that is bound to carboxyl groups in keratin proteins can transfer to other
binding sites over time [133].
Calcium(II) [129] and magnesium(II) have an affinity for the oxygen ligands of
carboxyl and carbonyl groups in amino acids [130] and the levels of these
metals, as well as copper and manganese, increase in wool after exposure to
sunlight [134]. Photo-oxidation increases the number of carbonyl species and
therefore increases the likelihood of the adsorption of metals into wool from
exogenous sources.
1.8.3. Distribution of metals in wool
The distribution of metals throughout wool and hair fibre is different for each
metal and overall the content is higher in the cuticle cells of wool than in the
cortex [5]. Metal analysis of separated cortical and cuticle cells has shown that
the levels of calcium and magnesium tend to be higher in the cuticle than in the
cortex, whereas iron, manganese and zinc are higher in the cortex and copper is
evenly distributed throughout the fibre [108]. A new technique using a
Synchrotron X-ray fluorescence microprobe has been used to image the trace
metals in black hair from a smelter worker. Copper, calcium and zinc were
distributed throughout the fibre, although more concentrated in the cuticle,
whereas iron was located only in the cuticle [124]. However, the distribution of
metals in coloured human hair is likely to be different from unpigmented wool
CHAPTER 1
22
because metals also have an affinity for melanins [135] and human hair is
frequently exposed to hair care products that can contain traces of metals.
Divalent cations from exogenous sources tend to accumulate at the scale edges
where they migrate into the hair, whereas monovalent cations bind to the flat
scale surfaces [129]. The tips of the wool fibre have a higher content of some
metals than the base of the fibre due to their greater exposure to the
environment and their higher affinity for water, although divalent cations can
migrate along the length of the fibre over time [120].
1.8.4. Measurement of trace metal content in wool
Hair and wool production cycles in three phases, which are growth (anagen),
regression (catagen) and rest (telogen) [136]. Like human hair follicles, which
have a two to six year growth phase, wool follicles also have a long anagen
period of at least two years [137]. Because of the long growth period and the
lack of seasonal variation, analysis of the trace metal content of human hair has
been used for biochemical monitoring of disease and environmental exposure.
The advantage of using hair and wool as a bioindicator is because of the
convenience of sampling and handling. The disadvantages are that normal
concentration ranges in human hair have not been established and that the level
of trace metals depends on variables such as age, sex, hair colour [138] and
adsorption from the environment. Compared to human hair, the trace metal
content of wool is more likely to be influenced by seasonal changes in diet,
climate and exposure to the environment. Another disadvantage is that a
standard method for preparing hair test specimens has not been developed and
there is little consensus on the appropriate washing regime prior to elemental
analysis [139].
Washing to remove unbound or occluded contaminants is essential if the trace
metal content of hair or wool samples is compared. Different washing
procedures will extract each metal to varying degrees [140-142]. For example
copper and zinc bind tightly to hair proteins and are only partially removed by
CHAPTER 1
23
prolonged washing in strong chelating agents [143] whereas iron and
aluminium are more easily removed using non-ionic detergent [144]. Therefore
it is essential to establish a reliable consistent method to wash test specimens.
Providing that the preparation and analyses are performed under strictly
controlled conditions, analysis using instrumental techniques such as
Inductively Coupled Plasma Mass Spectrometry (ICP-MS) [145], Neutron
Activation Analysis (NAA) [146] and Inductively Coupled Plasma Atomic
Emission Spectroscopy (ICP-AES) [147] ensure a reproducible comparative
analysis of trace metals in hair and wool.
1.9. SUMMARY
The literature reviewed in this chapter outlines the known mechanisms and
products associated with the photoyellowing and weathering of wool, the
mechanism of metal-catalysed oxidation of proteins and lipids in biological
systems and the processes whereby trace metals are incorporated into the wool
fibre. This chapter also introduces methods for measuring wool colour and
photostability, and the trace elemental analysis of keratin proteins. The
introduction cites existing evidence from the literature that variations in trace
metal content can affect wool photostability and wool colour.
This thesis proposes that the metals that are bound to the proteins in wool fibre,
particularly iron and copper, can catalyse the production of hydroxyl radicals in
photo-irradiated wool, and that intrinsic levels of these metals found in wool
fibre increase the rate of photoyellowing, since only trace levels of metals are
required for metal-catalysed oxidation of proteins [148]. The study will focus
on wet wool since Fenton-type reactions can only occur in an aqueous
environment. We hypothesise that the degree of metal-catalysed oxidation of
wool, and hence yellowness, is proportional to the concentrations of iron and
copper in the wool. However, several photo-oxidation processes may occur
simultaneously and different concentrations of absorbing amino acids and
species that may quench hydroxyl radicals before they damage wool may make
CHAPTER 1
24
the process more complex. Trace metals can also catalyse the oxidation of
lipids to form peroxides that then damage proteins, however, wool lipids are 1-
2% of the total mass of greasy wool and their effect on photo-oxidation on
wool proteins is likely to be negligible compared to the catalytic effect of
metals bound protein. This mechanism is not within the scope of this study.
Two reliable and reproducible test methods were developed in order to study
the role of trace metals on the photostability of wool fibre because there were
no standard methods available at the outset of the research. Development of a
protocol to clean and prepare a large number of wool samples for trace metal
analysis is described in Chapter 2 and the development of a method to measure
the photostability of small fleece wool samples is described in Chapter 5.
Chapter 3 outlines a study on the relationship between trace metal content with
photoyellowing, hydroxyl radical and carbonyl production in photo-irradiated
metal-doped wool fabrics. The effect of a selection of antioxidants and
chelating agents on the production of hydroxyl radicals in metal-doped fabric is
discussed, as well as the effect of fabric pH and removing the internal wool
lipids.
The association between trace metals and wool colour was evaluated using
samples taken from sheep in the Sheep CRC Information Nucleus Flock and is
described in Chapter 4. Studies of trace metals in wool and hair of livestock are
limited and are mainly related to assessing the trace metal status and nutrition
of animals [149] [150] [108], the effect of trace metals on the mechanical
properties of wool [146], assessing the effectiveness of different washing
procedures for preparing sample for trace metal analysis [142] and evaluating
the correlations between metal content and fibre characteristics such as the
colour of pigmented wool [151] and the onset of canary yellowing [122]. There
have not been any studies examining the influence of trace metals on the colour
and photostability of unpigmented wool.
A sub-set of the whitest wools analysed for trace metal content was selected to
study the effect of the intrinsic trace metals in wool fibre on the rate of
CHAPTER 1
25
photoyellowing after exposure to UV light. The results of this study are
discussed in Chapter 6.
Finally, the extent of light penetration through a Merino fleece was determined
using a simulated fleece. This study established the regions of a fleece that are
most vulnerable to photo-damage from exposure to solar radiation and
compared this to the trace metal content and the extent of damage. This study
is discussed in Chapter 7.
This study is part of the Whiter Lightfast Wool program of the Co-operative
Research Centre for Sheep Industry Innovation and builds on and contributes
to understanding the significance of metal-catalysed oxidation in keratin
proteins, the mechanisms and factors associated with wool photoyellowing,
and the correlations between trace metal content and the yellowness of wool. It
is anticipated that the knowledge attained from this study will provide
additional information to Australian wool growers and wool processors on how
to achieve whiter and more lightfast wools.
.
CHAPTER 2
26
CHAPTER 2. DEVELOPMENT OF A PROTOCOL TO PREPARE WOOL SAMPLES FOR TRACE METAL ANALYSIS
2.1. INTRODUCTION
Both wool and human hair are keratin fibres that are chemically similar,
therefore the same protocols to determine the trace metal content in human hair
can be used on wool, fur and animal hair. Although hair can provide a history
of trace metal status during growth, and samples are easy to collect, handle and
analyse, there is some degree of scepticism regarding the value of hair analysis
for medical diagnosis, largely because it is likely to be contaminated with hair
care products and there are no established normal levels of trace metals in
human populations. More importantly, there are no standard methods for
preparing samples [152] and there is considerable variability of results between
laboratories [153]. However, trace metal analysis of hair is considered valid if
conducted using strictly controlled analytical practices [154].
There are two main considerations when preparing wool or hair samples for
trace metal analysis. Firstly, the technique should not introduce any sources of
contamination or loss of material that could introduce errors. All laboratory
ware must be free of metal contaminants that may have been introduced during
manufacturing or during prior use. After thorough cleaning, the materials used
to manufacture the laboratory equipment should not leach, absorb or bind metal
analytes. Secondly, the test specimens must be rigorously cleaned in order to
remove residual contaminants and any unbound metal ions. Inconsistency
between laboratories in the trace metal analysis of human hair is partially due
to variations in sample preparation techniques or analyses carried out without
using adequate and reproducible cleaning methods [140]. Ideally, the washing
procedure should only remove external contaminants from the wool fibres
while retaining the endogenous trace metals. In practice, there is no guarantee
that washing completely removes all exogenous metal ions or that some
endogenous metal ions are not removed.
CHAPTER 2
27
Once cleaned, samples are ashed or digested to remove or solubilise the
organic material and release the metals. The most common method for
digestion of hair is refluxing in concentrated nitric acid with an oxidising agent
such as hydrogen peroxide or perchloric acid [141, 155-157] using either a
microwave oven [158] or a hot block. The trace metal content of the
hydrolysates can be analysed using instrumental techniques such as inductively
coupled plasma atomic emission spectroscopy (ICP-AES), or for more
sensitivity, using inductively coupled plasma mass spectroscopy (ICP-MS).
Due to the variability in sample collection and preparation methods and
analytical techniques, these protocols must be described in detail when
reporting trace metal contents. When measures of quality control and method
validation are taken to ensure consistency, and that each test specimen is
sampled and analysed in the same way, analysis results can be compared [154].
This Chapter outlines the development of a protocol to prepare wool samples
for quantitative determination of trace metals using ICP-AES. The method was
specifically developed for processing large numbers of wool samples from the
IN Flocks, the outcomes of which are discussed in Chapter 4.
2.2. MATERIALS AND METHODS
2.2.1. Plastic Laboratory Ware
During the initial work, the washing and digestion of wool samples was carried
out in glassware that was cleaned using 5% nitric acid. However, to eliminate
the requirement for repeated decontamination of laboratory glassware,
disposable plastic materials were later adopted for all laboratory ware used for
washing and digestion and were discarded after a single use. Elemental
analysis of plastic materials by neutron activation analysis determined that
conventional polyethylene and polystyrene are pure plastics. These materials
leach extremely low amounts of trace metals (~1 ng/cm3 ) [159] which is only
relevant when determining trace metals at ultra-low levels. Polypropylene
digestion tubes and accessories (Environmental Express, South Carolina, USA)
CHAPTER 2
28
are certified to have low levels of metals and are specifically designed for trace
metal analysis. Sterile polypropylene or polystyrene centrifuge tubes (50 ml)
were selected for the washing process because they are cost effective,
disposable and have double-sealed caps to prevent leakage when inverted.
2.2.2. Preparation of a control wool sample
A ram’s fleece (purchased from Lance Mann, Newtown, Victoria, Australia,
3220, MFD 19.4 m) was used as a control wool for the development of the
method for trace metal analysis, as a quality control standard for each
analytical run and for the development of a method to measure the colour and
photostability of small loose wool samples.
The tips of the wool were removed and the wool was scoured using the method
developed for maximising whiteness [160]. Wool samples (~10 g) were
scoured with Hydrapol TN450 (nonylphenol ethoxylate, Huntsman, Australia)
in distilled water using the conditions outlined in Table 2.1. This method was
developed to minimise residual wax and to produce optimal wool colour after
scouring and is not used for processing lots in industry. The dried, scoured
wool was processed through a Shirley Analyser twice to tease out the fibres
and to remove the majority of the vegetable matter.
Bath
Number
Chemical addition (ml) Total volume
of liquor
Temperature
(oC)
Dwell time
(min) 0.5%
detergent
0.1M
NaOH
1 180 15 250 65 3
2 70 4 300 60 2
3 20 4 400 50 2
4 20 0 300 60 2
5 0 0 400 50 2
Table 2.1 Scouring conditions for maximum whiteness.
CHAPTER 2
29
2.2.3. Cleaning and drying
A previous study confirmed that the copper, iron, manganese zinc and
cadmium content in hair samples did not change after four wash / rinse cycles
in acetone, an EDTA solution and anionic and non-ionic detergents [140].
Therefore the following method was adopted for cleaning wool in preparation
for trace metal analysis. Approximately 0.5g of scoured wool samples were
cleaned in four 20 minute wash / 20 minute rinse cycles with 30 ml of an
anionic detergent solution prepared with ultra-purified water (18.1 MΩ-cm).
The detergent solutions were 1% w/w sodium lauryl sulphate (SLS) and 1%
w/w Lissapol TN450 (nonylphenol ethyoxylate, ICI) and were prepared with
ultra purified water. Lissapol TN450 can be interchanged with Hydrapol
TN450 (Huntsman, Australia) since they are identical products.
To allow up to 40 samples to be processed simultaneously, wool was washed in
centrifuge tubes and agitated using variable speed suspension mixers. The
detergent and ultra-purified water were dispensed using reagent dispensers.
After cleaning, samples were rinsed four times with 30 ml of ultra-purified
water for 20 minutes each rinse. The caps of the centrifuge tubes were suitable
containers for washed wool when drying at 80oC overnight and cooling in a
desiccator.
2.2.4. Doping wool fibre
Wool fibre was cleaned with non-ionic surfactant according to the washing
regime described. The cleaned fibre was soaked for one hour at room
temperature in 100 ml of 30 ppm solutions of divalent copper (pH 5.1) iron
(pH 2.8), manganese (pH 5.8) and zinc (pH 5.4) sulphate salts that were
prepared in ultra- purified water. The iron salt solution was acidified to prevent
rapid oxidation of Fe2+
to Fe3+
. Half of each doped sample (~0.5 g) was
thoroughly rinsed in metal-free water and the other half was washed again
using the cleaning regime specified (2.2.3). The samples were thoroughly
dried and cooled in a dessicator.
CHAPTER 2
30
2.2.5. Digesting wool
There are many methods for solubilising proteins for trace metal analysis. Dry
ashing by charring samples at 1000°C is suitable for analyses of zinc, copper
and manganese, whereas iron may volatilise under these conditions [161]. The
most common method for preparing biological samples for trace metal analysis
is wet ashing in acid, and this is carried out most effectively using nitric acid
[162] together with an oxidant such as perchloric acid or hydrogen peroxide.
The measured sulphur content of hair indicates the effectiveness of the
digestion process, as low levels of sulphur are evidence that the heavily
crosslinked regions of the fibre have not been fully oxidised in the digestion
process [163].
Initially, wool samples were digested by refluxing with 6M HCl in Pyrex flasks
fitted with condensers. The residue build up in the flasks was removed after two or
three digestions by refluxing with clean hydrochloric acid for several hours.
This digestion system was clearly unsuitable for processing large numbers of
samples from the IN flocks. The Environmental Express
HotBlockDigestion System was adopted because 54 samples can be
processed simultaneously and the system employs polypropylene digestion
tubes and reflux caps that are disposable and free of trace metals. A mixture of
nitric acid and hydrogen peroxide is more effective at cleaving peptide bonds
[162] and was used instead of HCl.
Accurately weighed, 0.5g of clean and dried wool samples were digested in
polypropylene digestion tubes fitted with polypropylene reflux caps. The
reaction of the nitric acid/hydrogen peroxide/keratin mixture is exothermic and
the following digestion regime was employed to avoid the risk of effervescence
and loss of material. Samples were refluxed in 5 ml of 69.0% nitric acid
(TraceSELECT, Fluka) for 2 hours at 30°C, after which 2 ml of 35% hydrogen
peroxide (EMPROVE, Merck) was added. When the effervescence subsided,
the tubes were shaken and the temperature was raised to 115°C for 2 hours.
CHAPTER 2
31
The hydrolysates were cooled then diluted with ultra-purified water to 50 ml
using the moulded-in graduations on the digestion tubes which the
manufacturers claim to accurately measure volumes to within 0.5%. The
accuracy of the 50 ml graduation was confirmed by taking five independent
measurements of the mass of water using five digestion tubes. The mean of the
25 readings was 49.88 gm and the sample variance was 0.031 (0.24% error).
The solutions were filtered using 2m Teflonfaced polypropylene filters
(Environmental Express FilterMatewhich remained in the tubes. The
concentration of nitric acid in the test solutions is within the acceptable limit
for ICP-AES to prevent a loss of sensitivity.
2.2.6. Trace metal analysis
Wool hydrolysates were diluted to give a final acid concentration of 5% nitric
acid and the trace metal content was quantitatively determined by CSIRO
Process Science and Engineering (ex CSIRO, Minerals) using a Varian Vista
inductively-coupled plasma /atomic emission spectrometry (ICP/AES). In this
process, the test solutions are nebulised then introduced into the argon plasma.
The analytes emit radiation at specific wavelengths which allows
characterisation of several elements simultaneously. The calibration standards
were prepared with the same nitric acid matrix as the test solutions and
multiple calibration standards were required for analysis of the major and
minor elemental components.
2.3. RESULTS AND DISCUSSION
2.3.1. Comparison between cleaning methods
Wool and hair must be thoroughly cleaned in preparation for trace metal
analysis to ensure consistency. There is considerable variation in washing
regimes [140, 155] and washing solutions include organic solvents such as
ethanol, ether, acetone, hexane and carbon tetrachloride, detergents, acids and
chelating agents and each will extract metals to varying degrees
[155].
Chelating agents such as ethylenediaminetetraacetic acid (EDTA) and dilute
CHAPTER 2
32
hydrochloric and nitric acid should be avoided because they are likely to
remove some of the bound metals that are of interest to the analyst [164].
While there appears to be little consensus over the appropriate washing
procedure for trace metal analysis of human hair, an extensive wash in mild
detergent has been found to be suitable [141].
There was little difference in the trace metal content of wool fibre after four
washes with anionic and non ionic detergents (Table 2.2). Anionic detergents
containing sodium are unsuitable when determining metal content by neutron
activation analysis. High levels of sodium contaminate the wool creating a
large sodium signal in the spectrum after exposure to a neutron flux that
masks other elements. The non-ionic surfactant, Lissapol TN450, is widely
used in industry for scouring wool and is considered to be the most effective
detergent for removing wool wax [165]. This surfactant was used for this study
although other non-ionic detergents such as Triton X-100 (polyethylene glycol
octylphenyl ether) [156], 7X O-Matic (diethylene glycol monobutyl ether)
[144, 166] and Snoop (propylene glycol) [143] have been used as cleansing
agents in preparation of trace metal analysis of hair and are recommended as
acceptable.
Wash method Ca Cu Fe Mg Mn S Zn
unwashed mean 794 13 30 214 3.3 33600 120
sem 9.5 0.5 0 0.5 0 800 2.0
anionic detergent mean 123 10 9 10 0.7 34400 118
sem 19 0.4 0.5 0.5 0.05 300 2.0
non ionic detergent mean 139 11 9 15 1.1 33300 116
sem 17.5 1 1 1.5 0.2 400 0
non ionic detergent +EDTA mean 86 5 8 11 0.3 33050 21
sem 16 0.35 0.5 1.5 0 50 0.5
non ionic detergent+ OA mean 376 8 24 6 0.3 32650 15
sem 6 0.3 0.5 0.5 0.05 250 1.5
Table 2.2 Trace metal content (mg/kg), mean and standard error of the mean (sem)
of duplicate samples of scoured unwashed wool and scoured wool cleaned with four
washing regimes prior to digestion with anionic and non-ionic detergent and non ionic
detergent with ethylene diamine tetraacetic acid (EDTA) and oxalic acid (OA) .
CHAPTER 2
33
Copper binds tightly to wool and is only partially removed by extended
washing in oxalic acid and EDTA (Table 2.2). However, ashing pre-cleaned
metal-doped wool in non-ionic detergent solutions can extract approximately
30% of copper and iron and almost 80% of manganese whereas only 13% of
zinc is removed (Table 2.3).
doping metal Wash method Cu Zn Fe Mn
copper rinse 75 116 14 0.2
non ionic detergent 53 114 12 0.1
zinc rinse 8 163 17 0.3
non ionic detergent 10 142 17 0.3
iron rinse 8 72 30 0.4
non ionic detergent 8 38 20 0.2
manganese rinse 9 142 22 18
non ionic detergent 8 122 18 4
Table 2.3 Trace metal content (mg/kg) of pre-cleaned wool doped with copper, zinc,
iron and manganese then either rinsed with metal-free water or washed with non-ionic
detergent.
2.3.2. Quality control procedures
Several procedures were carried out to determine the reliability and
repeatability of the method. Firstly, analysis of a blank solution of nitric
acid/hydrogen peroxide refluxed in digestion tubes confirmed that materials
and reagents used in the digestion process do not introduce sources of trace
metal contamination (Table 2.4).
Secondly, the accuracy of the ICP-AES was evaluated by analysing a certified
multi-element spectroscopy standard. With the exception of chromium and
nickel, which are not of interest in this study, the accuracy for the elements
analysed was within 1 mg/kg.
Thirdly, standard hair samples obtained from the International Atomic Energy
Agency (Vienna) were used as a reference to determine the reliability of the
digestion and ICP-AES method (Table 2.4). The difference between the
certified levels of trace metals and the measured value was no greater than
0.24% and for most metals less than 0.1%.
CHAPTER 2
34
Ca Cu Fe Mg Mn S Zn
Blank 0.1 <0.05 0.01 <0.05 <0.1 <50 0
multi-element standard
Measured value 9.9 9.5 9.3 10.7 9.3 0.2 9.8
Expected value 10 10 10 10 10 0 10
% difference -0.01 -0.05 -0.07 0.07 -0.07 -0.02
IAEA-085 standard hair
Measured value 912 17 64 129 8.5 48000 158
Expected value 929 16.8 79.3 140 8.8 163
% difference -0.02 0.01 -0.19 -0.08 -0.03 -0.03
IAEA-086 standard hair
Measured value 1220 18 94 177 10 48400 168
Expected value 1120 17.6 123 177 9.6 167
% difference 0.09 0.02 -0.24 0 0.04 0.01
Table 2.4 Trace metal content (mg/kg) of a blank solution of nitric acid/hydrogen
peroxide refluxed in polypropylene digestion tubes, a multi-element spectroscopy
standard and IAEA-085 and IAEA-086 Standard Hair.
Finally, ten scoured wool samples from the same sheep were used as a control
and were processed in ten independent experiments to estimate the
repeatability of the entire process of sampling, washing, digestion and analysis
(Table 2.5). The repeatability of the analyses was estimated from the relative
standard deviation (RSD) values and the Horwitz Ratio for intra-laboratory
precision (HorRat(r)) [167]. HorRat(r) is calculated by dividing the % relative
standard deviation of the replicates (RSD) by the predicted relative standard
deviation (PRSD) (Figure 2.1).
Figure 2.1 Horwitz Ratio equation for determining inter and intra laboratory
precision of analytical measurements.
HorRat(r) =RSD
PRSD HorRat(r) =
RSD
PRSD
RSD is the % relative standard deviation of the replicates
PRSD = 2C-0.15 is the predicted relative standard deviation where C is the
concentration of the analyte expressed as a mass fraction
CHAPTER 2
35
The acceptable limits of HorRat(r) values for intra-laboratory validation is 0.25
to 1.34. Only iron, sulphur and zinc were within acceptable limits. However,
with the exception of magnesium and possibly manganese, all analytes had
acceptable HorRat(R) values for inter-laboratory variability which must be
between 0.5 and 2.0.
The wool used as a control was sampled from different parts of the entire
fleece and several analyses were not within acceptable limits. It is possible that
the variation in analytical results could be partially attributed to inadequate
blending of the fibres during the scouring and combing process (Table 2.5).
The method described in this chapter was used for mid-side wool samples that
were scoured and blended at AWTA (Chapter 4). Variation between replicates
due to incomplete blending is likely to be less for these samples.
Test Number Ca Cu Fe Mg Mn S Zn
1 133 7.5 9 14 0.2 36000 120
2 112 7.8 9 14 0.1 35900 136
3 117 8 9 13 0.2 35500 129
4 127 7.5 8.8 21 0.5 35600 123
5 96 5 7.7 9 0.4 35500 122
6 106 5 8 10 0.4 37000 129
7 97 5 8 8 0.4 35300 122
8 120 4.9 8.1 13 0.4 35800 130
9 120 5 10 15 0.5 35100 129
10 104 4.8 9 11 0.4 33800 126
Mean 113 6.1 8.7 13 0.4 35550 127
RSD 11.0 23.6 8.1 28.9 38.7 2.3 3.9
HorRat (r) 1.4 1.9 0.7 2.7 2.1 0.7 0.5
Table 2.5 Trace metal content (mg/kg) of 10 independent analyses of 10 sub-
samples of the control wool. RSD = s/x × 100 where s is the standard deviation and x
is the mean.
The developed method was used to determine the trace metal content of 117
duplicate test wool specimens. The duplicate analyses were averaged and the
difference between the measured value and the mean value of the two analyses
was calculated for each result to determine the “within-sample residuals”. The
CHAPTER 2
36
standard deviation of these values was calculated for each analyte as an
estimate of the overall variation between duplicate analyses (Table 2.6).
Although there is greater variability in replicate analyses of calcium and
sulphur, the differences in duplicate analyses of copper, iron, magnesium,
manganese and zinc were low and are acceptable.
Element
Between sample
standard
deviation
Standard deviation of
the within sample
residuals
Calcium 202 24.5
Copper 1.5 0.3
Iron 10.5 2.2
Magnesium 24.2 3.7
Manganese 2.1 0.2
Sulphur 2342 771
Zinc 16.5 2.3
Table 2.6 The standard deviation of the samples and the variation between
duplicates within samples calculated from the standard deviation of the differences
between the measured value, and the mean value of duplicate analyses of 117 wool
samples.
2.4. SUMMARY
A regime for washing and digesting fleece wool samples in preparation for
trace metal analysis by ICP/AES was developed using the Environmental
Express HotBlocksystem. This protocol allows timely processing of large
numbers of samples and the use of disposable centrifuge tubes, digestion tubes
and filters is convenient, cost efficient and reduces the risk of cross-
contamination.
Whilst it cannot be guaranteed that the washing procedure only removes
unbound metals while retaining the bound metals, the trace metal content of
samples can be compared when they are prepared and analysed under the same
conditions. The procedure outlined in this chapter was used to determine the
trace metal content of fibre samples from sheep in the IN flocks and the results
are discussed in Chapter 4.
CHAPTER 3
37
CHAPTER 3. OXIDATION AND YELLOWING IN PHOTO-IRRADIATED METAL-DOPED WOOL
3.1. INTRODUCTION
Iron and copper are considered to be the most important catalysts for the
oxidation of proteins in biological systems [81]. These metals form complexes
with proteins and in the presence of a reducing agent, can react with hydrogen
peroxide to produce hydroxyl radicals. However, other metals that can undergo
single electron transfer, which include most fourth period transition elements
[168], can catalyse the production of hydroxyl radicals via Fenton-type
reactions in the presence of water. Under certain conditions, nickel [169],
manganese [170], vanadium [171] and cobalt [172] are involved in the
generation of reactive oxygen species and the oxidation of amino acids.
Protein-bound iron and copper also increase free radical production,
photoyellowing [6] and the photo-sensitivity of dry wool [5]. The production of
hydroxyl radicals, which are generated when wet wool is exposed to UV,
decreases with the addition of an iron chelator [44]. The suggested mechanism
of metal-catalysed production of hydroxyl radicals proposes that chromophores
in the wool absorb UV wavelengths and react with oxygen to form hydrogen
peroxide via the superoxide anion. Hydrogen peroxide can react with metal
ions to produce hydroxyl radicals that oxidise amino acid residues in proteins
[44] (Scheme 1).
Scheme 1 Proposed mechanism for metal-catalysed oxidation of wet wool [44].
Chromophore + h Chromophore* (1)
Chromophore* + O2 Chromophore* + O2 ¯ (2)
2O2¯ + 2H+ H2O2 + O2 (3)
H2O2 + Mn+ M(n+1)+ + OH + OH¯ (4)
M(n+1)+ + O2¯ Mn+ + O2 (5)
CHAPTER 3
38
Hydroxyl radical production can be assayed using terephthalic acid (TA) under
alkaline conditions. The terephthalate anion reacts specifically with hydroxyl
radicals to form hydroxyterephthalic acid (HTA) that is detectable using
fluorescence spectroscopy [44, 173, 174]. The assay method is sensitive but not
quantitative because hydroxyl radicals are highly reactive and short-lived and
can react with species other than TA in protein systems. The fluorescence
intensity of HTA is linearly correlated to concentrations less than 35M,
making TA a suitable probe to monitor the production of hydroxyl radicals in
photo-irradiated wool [44].
Oxidation of the amino acid residues in wool after exposure to UV results in
the production of carbonyl species [89] and increased levels of amides [61].
Consequently, the carbonyl content of proteins is one indicator of the degree of
protein oxidation mediated by reactive oxygen species [175]. The production
of carbonyl species in proteins can be quantified by derivitisation with 2,4-
dinitrophenylhydrazine (2,4-DNPH) [50, 176] and the concentration of the
protein-bound hydrazone can be determined by colorimetric analysis [175].
Using this method, the carbonyl content has been shown to increase in photo-
irradiated wool yet there is no direct correlation with the extent of
photoyellowing [50]. It is not known whether trace metals within wool are
involved in the mechanism of carbonyl production.
The aim of this study was to determine which metals in fleece wool were likely
to act as catalysts for the oxidation of proteins and yellowing in photo-
irradiated wool. The role of trace metals was examined by quantifying
hydroxyl radical and carbonyl production and the extent of photoyellowing in
metal doped wool after exposure to UV. The effect of antioxidants, metal
chelators, pH on hydroxyl radical production was also investigated, as well as
the removal of wool lipids because light can induce the production of lipid
peroxy radicals [97] via metal-catalysed reactions. Although the level of lipids
in the wool fibre is low (1.0-1.5% on weight of wool) [177], their effect on
hydroxyl radical production is of interest because they could influence the rate
of protein oxidation. This study also examined the role of copper and iron on
CHAPTER 3
39
the extent of damage and loss of fibre strength from exposure to an artificial
light source.
3.2. MATERIALS AND METHODS
3.2.1. Preparation of fabrics
3.2.1.1. Metal-doped wool
The fourth period transition metals usually present in fleece wools are copper,
iron, manganese and zinc, whereas very little cobalt, nickel and vanadium are
detected [108]. For that reason, plain weave wool fabric was doped with
manganese, copper, zinc and iron by wetting out with a non-ionic detergent
(Leophen M, 0.1%) and shaking in approximately 2.5, 5, 10, 20 and 50 ppm
metal salt solutions that were prepared with ultra-pure water (Barnstead Water
Purification system). The iron salt solution was acidified with sulphuric acid
(to pH 2) to prevent rapid oxidation of Fe2+
to Fe3+
. Other metal salt solutions
were prepared without pH control (pH< 4). Samples were treated for 30
minutes at a liquor ratio of 3:1 and the excess metal salt solution was removed
by rinsing the doped wool in approximately 100 ml ultra-pure water, draining,
then shaking in a fresh bath of 100 ml of ultra-pure water for 15 minutes.
A control fabric (untreated) was prepared by soaking and rinsing fabric in
ultra-pure water for the same period of time.
3.2.1.2. Lipid-depleted wool
Scoured wool fabric has little wool grease remaining on the external surfaces
of the fibre and the majority of the internal lipids in wool were removed from
wool fabric by Soxhlet extraction in chloroform/methanol (79:21) for four
hours.
CHAPTER 3
40
3.2.1.3. Adjustment of fabric pH
Fabric was soaked overnight in 0.4M phosphate buffer solutions (pH 3.3, 4.7,
6.3, 7.6, 8.5 and 9.7) prepared in ultra pure water at a liquor ratio of 50:1. The
samples were centrifuged and air dried.
3.2.2. Trace metal content of fabrics
Without further washing, untreated, metal-doped and lipid-depleted wool
samples were dried and the trace metal content was determined using the
digestion and analysis method described in Chapter 2. The analysis results are
shown in Table 3.1.
Sample
Number
Concentration
of doping
solution
(ppm)
Copper Iron Manganese Zinc
untreated ― 4.1 29 4.2 119
Cu 1 2.5 22 28 4.1 114
Cu 2 5 42 28 4.1 117
Cu 3 10 73 28 3.8 115
Cu 4 20 154 29 3.7 113
Cu 5 50 283 29 3.1 108
Fe 1 2.5 3.8 42 4 112
Fe 2 5 3.9 53 3.6 110
Fe 3 10 4.1 77 3.5 112
Fe 4 20 4.3 90 3.2 111
Fe 5 50 4.1 78 2.7 104
Mn 1 2.5 3.8 28 16 113
Mn 2 5 3.7 26 26 109
Mn 3 10 3.7 28 50 113
Mn 4 20 3.7 27 81 114
Mn 5 50 3.6 25 123 108
Zn 1 50 4.9 28 3.6 312
lipid depleted ― 4.2 32 4.8 124
pH 3.3 ― 3.3 45 0.1 4.5
pH 7.6 ― 4.6 109 1 95
pH 9.7 ― 4.8 39 2 105
Table 3.1 Trace metal content (mg/kg) of untreated, metal-doped, lipid depleted wool
and pH adjusted wool.
CHAPTER 3
41
These results show that wool has a greater affinity for zinc and copper ions
compared to either iron or manganese and that the uptake of manganese is
greater than iron.
3.2.3. Determination of hydroxyl radical production in wool
Hydroxyl radical production was determined using the method described
previously [44]. Untreated and metal-doped fabric samples (10 x 30 mm) were
placed against the inside transparent surface of PMMA cuvettes and 3.2 ml of a
freshly prepared solution of TA (2 mM) in a 50 mM phosphate buffer (pH 7.6)
was added. For each sample, a similarly treated fabric that was not irradiated
was used as a control. The cuvettes were placed in direct contact with a Philips
TUV tube (UVA) (Figure 3.1) for one hour after which the fabric samples were
immediately removed from the cuvettes. Blanks were prepared with irradiated
and unirradiated solutions of TA. A UVB light source is not suitable for this
assay because TA strongly absorbs 250-280 nm wavelengths. The rate of
oxidation of metals in Fenton-type reactions is dependent on the pH and greater
levels of oxidation occur at higher pH [178]. Therefore, the alkaline conditions
of the hydroxyl assay do not inhibit these reactions.
The production of HTA was measured (ex=315nm, em=425nm) using a
Hitachi Model 4500 fluorescence spectrophotometer. Irradiated samples were
tested in duplicate and the results were averaged. The fluorescence intensity
was corrected using the unirradiated control samples.
Solutions of phosphate-buffered TA were also prepared with 0.1 mM of
antioxidants; ascorbic acid (AA) and citric acid (CA) and chelating agents;
deferoxamine mesylate (desferal), diethylenetriamine pentaacetic acid (DTPA)
and ethylenediamine tetraacetic acid (EDTA).
CHAPTER 3
42
Figure 3.1 Spectral intensity of Philips TUV (UVA) lamp.
3.2.4. Irradiation of fabric to assess photoyellowing
Fenton-type reactions require an aqueous medium, therefore all wool samples
were prepared by soaking in a surfactant solution (0.1% Leophen M),
removing the excess fluid and sealing in plastic bags. The bags were placed
directly in contact with a broadband UVB fluorescent tube (Philips
TL20W/12RS) and irradiated for four hours after which the samples were
removed from the bags and air dried.
The spectral output of the UVB light source has a maximum intensity at
approximately 300 nm and the major peak ranges from 280–320 nm (Figure
3.2) and the UVA lamp has a spectral output ranging from 350-400 nm with
the maximum intensity occurring at 365 nm (Figure 3.1). The UVA and UVB
lamps both produce wavelengths which cause wool to photoyellow. The
spectral intensities of the light sources were measured using an AvaSpec-3648-
USB2 spectrometer fitted with a FC-UV200 200 µm diameter fibre-optic cable
probe (Avantes BV, Erbeek, Netherlands).
0
50
100
150
200
250
300
200 240 280 320 360 400 440 480 520 560 600
Irra
dia
nce
[µ
Wa
tt/c
m²]
Wavelength (nm)
CHAPTER 3
43
Figure 3.2 Spectral intensity of Philips TL20W/12RS (UVB) lamp.
3.2.5. Determination of carbonyl groups in wool
Irradiated and unirradiated metal-doped wool samples were conditioned to
constant weight at 20oC and 65% relative humidity. Snippets of accurately
weighed wool (~10 mg) were mixed with 2M HCl (0.5 ml) in digestion vials.
The vials were placed under vacuum for 10 minutes to degas the HCl then
purged of oxygen by streaming nitrogen over the liquid for approximately 2
minutes. After sealing with gas-tight lids, the vials were heated in an oven at
80oC for 18 hours then cooled to room temperature.
The method used to determine the carbonyl content of wool has been described
previously [50]. Aliquots (0.5 ml) of a solution of 2,4-DNPH (0.5 mM)
prepared in 2M HCl were added to vials containing digested wool and to a
blank containing 2M HCl (0.5 ml) only. The hydrazone derivative was allowed
to develop for 20 minutes at room temperature after which 2M NaOH (4.0 ml)
was added to develop the colour. A standard series was prepared using pyruvic
acid that was treated with 2,4-DNPH using the same procedure as the samples
and the blank. The absorbance of all solutions was measured at 500 nm against
0
10
20
30
40
50
60
70
80
90
200 240 280 320 360 400 440 480 520 560 600
Irra
dia
nce
[µ
Wa
tt/c
m²]
Wavelength (nm)
CHAPTER 3
44
the blank using a Varian Cary Bio 300 UV-Vis Spectrophotometer
(Melbourne).
3.2.6. Colour measurement
The colour of four layers of wool fabric was measured with a Gretag Macbeth
Color-Eye 7000A reflectance spectrophotometer (Munich, Germany) using the
small area view (SAV) aperture (7.5 x 10 mm), a D65 light source and a 10°
collection angle with the spectral component included (SCI). The CIE
tristimulus values X, Y and Z were measured by averaging three readings and
were used to calculate the wool yellowness (Y-Z).
3.2.7. Assessment of tensile strength
Dry samples of untreated and iron- and copper-doped wool, together with a
similar set of wet samples in sealed plastic bags, were weathered by exposure
to a mercury ballast tungsten filament (MBTF) phosphor-coated lamp for 672
hours [179]. The MBTF is commonly used for accelerated ageing and
colourfastness tests for textiles using Australian Standard 2001.4.21-2006. The
major emission peaks occur at 365 and 435 nm with minor peaks at 405, 550
and 580 nm (Figure 3.3).
Figure 3.3 Spectral intensity of Philips MBTF lamp.
0
50
100
150
200
250
300
350
400
450
200 300 400 500 600 700 800
Irra
dia
nce
[µW
att
/cm
²]
Wavelength (nm)
CHAPTER 3
45
Moisture permeated through the plastic bags and the wet samples had dried at
some point during the exposure period. The wet burst strength of irradiated
wool fabrics was assessed [180] by averaging the results of four measurements.
3.3. RESULTS AND DISCUSSION
3.3.1. Hydroxyl radical production in metal-doped wool
Of the metals trialled in this study, iron had the strongest effect on hydroxyl
radical production, the extent of which increased with iron content (Figure 3.4).
Although ferric and ferrous ions can form complexes with phosphate, the
greater production of hydroxyl radicals in iron-doped wool suggests that the
phosphate buffer used in this study did not inhibit the catalytic effect of iron.
Previous studies have also shown that oxidation of Fe2+
to Fe3+
generates
hydroxyl radicals in the presence of oxygen and a reducing agent [178] and
that iron-sulphur complexes in photo-irradiated wool proteins are likely to be
associated with the production of carbon-centred radicals on adjacent amino
acid residues [4].
Figure 3.4 Corrected fluorescence intensity of hydroxyterephthalic acid (HTA)
(ex=315 nm, em=425 nm) indicating hydroxyl radical production after irradiation
with UVA for one hour. Samples shown are TA without wool (TA), untreated wool
(Unt) and wool doped with different copper (Cu 1, Cu 2, Cu 5), iron (Fe 1, Fe 2, Fe 5)
and manganese (Mn 1, Mn 2, Mn 5) contents.
0
1
2
3
4
5
6
7
8
TA Cu 1 Cu 5 Fe 2 Mn 1 Mn 5
Flu
ore
sc
en
ce
In
ten
sit
y
Unt Cu 2 Fe 1 Fe 3 Mn 2 Zn 1
CHAPTER 3
46
The oxidation Cu+ to Cu
2+ can also catalyse hydroxyl radical production [178]
and low levels of copper in wool (4.8 mg/kg) increase the production of glycyl
radicals after photo-irradiation [5]. In contrast, this present study has shown
that the concentration of HTA was unaffected by the presence of copper.
Although thiol groups in wool are capable of reducing divalent copper, thereby
enabling it to act as a catalyst, it is possible that the copper complexes with
nitrogen and sulphur species in wool which stabilise Cu+
and reduces its
catalytic activity [178]. It is also possible that the extent of hydroxyl radical
production in copper-doped wool was similar to wool treated with iron, except
that hydroxyl radicals reacted with the amino acid residues close to the copper
binding sites instead of reaction with TA.
Zinc did not affect hydroxyl radical production because it is redox inactive and
therefore cannot act as a catalyst for oxidation of proteins. On the other hand,
the presence of manganese reduced the extent of hydroxyl radical production in
photo-irradiated wool. The anti-oxidant effect of manganese has been shown
previously from in vitro studies on rat’s brains [181]. Although unable to
quench hydroxyl radicals directly, some manganese (III) can inhibit hydroxyl
radical production by dismutating superoxide (reaction 1, Scheme 2) and
reacting with hydrogen peroxide to produce molecular oxygen (reaction 2,
Scheme 2) [55]. Manganese (II) complexes can also catalyse the dismutation of
superoxide [182]. Another possibility is that Mn (II) prevents iron from acting
as a catalyst by inhibiting the reduction of Fe3+
to Fe2+
[183].
Scheme 2 Reactions of manganese (III) with superoxide and hydrogen peroxide.
3.3.2. Chelating agents and antioxidants
Chelating agents that sequester metals and antioxidants that scavenge hydroxyl
radicals possibly protect proteins by reducing the effect of metal-catalysed
Mn+3 + O2¯ Mn+2 + O2 (1)
H2O2 + 2Mn+3 2Mn+2 + O2 + 2H+ (2)
CHAPTER 3
47
oxidation. Since metals influence the rate of radical production in wet wool
exposed to UV, it is highly probable that the same processes occur in fleece
wools which are exposed to sunlight and moisture. The application of
treatments to minimise the catalytic effect of trace metals could offer some
degree of protection to fleeces [134] and further investigation is warranted.
Desferal is an iron chelator that is used clinically to treat excesses of iron in the
body and it inhibits lipid peroxidation by hydroxyl radicals that are produced
by iron-catalysed reactions [106]. In this study and in previous work [44],
chelation with desferal prevents iron from acting as a catalyst for hydroxyl
radical production in photo-irradiated wool. It is likely that the small but
measurable concentration of HTA generated in untreated and copper- and
manganese-doped wool in TA is due to the presence of iron. Each of these
samples had an iron content of at least 25 mg/kg and the addition of desferal
effectively reduced HTA production (Figure 3.5).
The addition of DTPA also reduced the amount of hydroxyl radicals detected,
although to a lesser extent than desferal. Metal complexes require at least one
free co-ordination site to act as a catalyst for hydroxyl radical production [184].
The DTPA ligand can form eight co-ordination bonds which completely
encloses the metal ion, thus preventing its binding to proteins. In contrast,
EDTA acts as pro-oxidant in photo-irradiated wool and this effect has been
studied previously [81, 185], particularly in the presence of iron [186]. EDTA
forms six co-ordination bonds, therefore it is unable to completely surround the
metal ion [187] and it acts as a carrier of the metal to the protein [81]. This
mechanism is usually associated with proteins which have non-specific metal
binding sites, such as the proteins in wool. For proteins that have tight and
specific metal binding sites, EDTA sequesters metal ions, preventing them
from binding to proteins and acting as catalysts [183].
CHAPTER 3
48
Figure 3.5 Corrected fluorescence intensity of hydroxyterephthalic acid (HTA)
(ex=315 nm, em=425 nm) after irradiation of untreated and metal doped wool (Cu 5,
Fe 5 and Mn 5) with UVA for one hour in terephlalic acid (TA) with or without
ascorbic acid (AA), citric acid (CA), deferoxamine mesylate (desferal),
diethylenetriamine pentaacetic acid (DTPA) and ethylenediamine tetraacetic acid
(EDTA).
Ascorbic acid is a strong reducing agent which can also act as an antioxidant in
biological systems by interacting with more reactive radicals to form the
relatively unreactive ascorbate radical [188]. At low concentrations [184] and
under the conditions used in this study (pH 7, 50 mM phosphate), ascorbate
acts as a pro-oxidant in photo-irradiated wool. The ascorbate anion can auto-
oxidise to yield hydroxyl radicals but requires catalytic metals, even at low
levels, for the reaction to proceed [189]. Although solutions were prepared
with ultra-purified water, contaminants in the reagents and metals from the
doped fabric provided sufficient metal ions to generate hydroxyl radicals, as
shown by the large fluorescence peaks of the unirradiated samples (Table 3.2).
It is interesting to note that the concentration of HTA in unirradiated solutions
was higher in the copper/AA system. As a reducing agent, ascorbic acid assists
copper and iron to act as catalysts by recycling ferric and cupric ions and this
effect has been shown in this study, and previously [190]. Citric acid also
promotes the generation of hydroxyl radicals, although to a lesser extent than
EDTA and ascorbic acid.
0
20
40
60
80
100
120
TA TA/AA TA/CA TA/desferal TA/DTPA TA/EDTA
Flu
ore
sc
en
ce
In
ten
sit
y no fabric
untreated
copper
iron
manganese
CHAPTER 3
49
Fluorescence intensity
no wool fabric untreated Cu 3 Fe 3 Mn 3
TA 64 73 79 99 80
TA/AA 2343 2025 5991 2803 1825
TA/CA 38 75 81 114 82
TA/desferal 45 78 85 105 87
TA/DTPA 72 72 78 112 85
TA/EDTA 40 79 87 114 85
Table 3.2 Fluorescence intensity of hydroxyterephthalic acid (HTA) (ex= 315nm,
em= 425nm) produced by soaking untreated and copper-, iron- and manganese-doped
wool (Cu 3, Fe 3, Mn 3 wool fabric for one hour in solutions of terephthalic acid (TA)
with or without ascorbic acid (AA), citric acid (CA), deferoxamine mesylate
(desferal), diethylenetriamine pentaacetic acid (DTPA) and ethylenediamine
tetraacetic acid (EDTA). Solutions without fabric were used as controls.
It is likely that chelates which do not occupy all the co-ordination sites on iron,
leaving at least one site free, have a pro-oxidant effect on hydroxyl radical
production [191]. This effect has been confirmed in this present study with
photo-irradiated wool in the presence of EDTA, ascorbic acid and citric acid.
These reagents should therefore be used with caution when selecting anti-
oxidant and metal chelating treatments for wool.
3.3.3. Colour change after exposure to UVB
Although having a small effect on the initial yellowness (Table 3.3), doping
wool with copper and iron changed the colour of wool after exposure to UVB
for four hours while wet (Table 3.4). The change in colour is the result of
mechanisms that simultaneously create and destroy chromophores. In
particular, the brightness (Y) of iron-doped wool was reduced and was visibly
darker and duller than untreated wool, whereas the Y-Z values indicated that
the extent of yellowing was less (Table 3.3).
Yellowness (Y-Z) is used as an index of the colour of natural ecru fibre
however, this measure is not suitable for samples that are coloured or stained.
Therefore the colour of stained metal-doped fabric is best described by
differences in the reflectance spectra, or more conveniently by the X, Y and Z
tristimulus values.
CHAPTER 3
50
For photo-irradiated wool, an iron content of 42 mg/kg (Fe 1) and a copper
content of 22 mg/kg (Cu 1) were sufficient to induce a colour change and the
depth of shade increased with metal content. On the other hand, manganese and
zinc had little effect on the brightness before and after exposure to UV (Table
3.3).
sample before irradiation after irradiation 4hrs UVB
X Y Z Y-Z X Y Z Y-Z Y Y-Z
undoped 62.1 65.1 56.1 9.0 59.3 62.6 49.7 12.9 -2.5 3.9
Cu 1 61.6 64.7 56.4 8.3 58.4 61.6 47.9 13.7 -3.1 5.4
Cu 2 61.5 64.8 56.1 8.6 57.9 60.9 47.4 13.6 -3.8 4.9
Cu 3 60.4 63.6 54.7 8.9 56.9 59.7 45.5 14.3 -3.8 5.4
Cu 4 59.9 63.2 55.2 8.0 54.8 57.4 42.1 15.3 -5.9 7.2
Cu 5 58.8 62.4 54.4 8.0 51.2 53.3 37.2 16.0 -9.1 8.1
Fe 1 61.2 64.3 56.3 7.9 55.7 58.9 47.4 11.5 -5.4 3.5
Fe 2 59.8 62.7 54.8 7.9 53.2 56.4 46.4 10.0 -6.3 2.1
Fe 3 58.5 61.5 53.3 8.1 52.1 55.2 46.0 9.2 -6.2 1.0
Fe 4 58.6 61.6 53.8 7.8 50.5 53.5 44.3 9.2 -8.1 1.4
Fe 5 59.5 62.5 54.0 8.5 51.8 54.9 44.8 10.1 -7.6 1.6
Mn 1 61.7 64.7 55.6 9.1 58.4 61.7 47.9 13.8 -3.1 4.7
Mn 2 62.5 65.7 57.2 8.4 58.2 61.5 46.9 14.6 -4.2 6.1
Mn 3 62.5 65.7 57.2 8.4 58.8 62.0 47.9 14.1 -3.6 5.7
Mn 4 62.6 65.8 57.6 8.2 59.3 62.7 48.9 13.8 -3.1 5.6
Mn 5 62.4 65.6 57.0 8.6 59.2 62.6 48.7 13.9 -3.0 5.2
Zn 1 61.3 64.4 55.5 9.0 58.3 61.6 48.6 13.0 -2.8 4.0
Table 3.3 CIE tristimulus values and Y-Z of untreated and copper- (Cu 1-5), iron-
(Fe 1-5), manganese- (Mn 1-5) and zinc- (Zn 1) doped wool before and after exposure
to UVB for four hours while wet and the change in yellowness (Y-Z) and brightness
(Y).
Wool can be discoloured by boiling with as little as 0.5 ppm of iron in the wash
liquor [192] which is most likely due to the formation of iron complexes or
oxides. It is possible that these species are generated in metal-doped and photo-
irradiated wool. By removing all free and loosely bound metals with a strong
chelating agent (oxalic acid, 0.5%), the colour of irradiated wool after these
treatments is most likely from both the natural wool chromophores and those
induced by exposure to UV. Oxalic acid does not effectively remove bound
CHAPTER 3
51
iron (Table 2.2). Therefore, the Y-Z value can be used as an index of colour for
these treated wools.
All metal-doped wools were yellower after the oxalic acid treatment compared
to undoped wool. Overall, copper had the strongest effect on photoyellowing
(Table 3.4) suggesting that copper is not stabilised by thiols and is free to act as
a catalyst.
4hrs UVB and oxalic acid
treatment
compared to
unirradiated wool
compared to
irradiated wool
X Y Z Y-Z Y Y-Z Y Y-Z
undoped 64.7 68.4 55.4 13.0 3.3 4.0 5.8 0.1
Cu 1 63.4 67.0 51.6 15.4 2.3 7.1 5.4 1.7
Cu 2 63.0 66.5 51.1 15.3 1.7 6.7 5.5 1.8
Cu 3 61.7 65.0 49.2 15.9 1.5 7.0 5.3 1.6
Cu 4 60.4 63.6 46.9 16.7 0.4 8.6 6.2 1.4
Cu 5 57.7 60.6 44.2 16.4 -1.8 8.4 7.3 0.4
Fe 1 64.2 67.9 52.5 15.4 3.6 7.4 9.0 3.9
Fe 2 64.1 67.8 52.6 15.2 5.1 7.3 11.4 5.2
Fe 3 64.3 68.0 53.0 15.0 6.6 6.9 12.8 5.8
Fe 4 64.0 67.7 51.9 15.7 6.0 7.9 14.1 6.5
Fe 5 64.3 68.0 53.3 14.7 5.5 6.2 13.1 4.6
Mn 1 63.7 67.2 51.8 15.4 2.5 6.3 5.5 1.6
Mn 2 63.6 67.3 50.7 16.6 1.6 8.1 5.8 2.0
Mn 3 63.9 67.5 52.0 15.5 1.8 7.0 5.5 1.3
Mn 4 64.0 67.7 52.6 15.1 1.8 6.8 4.9 1.3
Mn 5 64.1 67.7 53.1 14.6 2.1 5.9 5.1 0.7
Table 3.4 CIE tristimulus values and Y-Z of untreated and (Cu 1-5), iron (Fe 1-5)
and manganese (Mn 1-5) doped wool after exposure to UVB for four hours while wet
and removing unbound metals with oxalic acid (0.5%).
Treatment with oxalic acid increased the brightness of all wools and the Y
value of treated iron-doped wool was similar to undoped wool. Therefore,
some of the colour change in metal-doped and photo-irradiated wool is due to
the production of coloured species that are not strongly bound to wool proteins.
CHAPTER 3
52
Acidifying wool has been shown to reduce the rate of photobleaching, thereby
increasing the rate of photoyellowing in sunlight [193]. However it is unlikely
that this phenomenon would account for differences in photoyellowing in this
present study. Wool was doped with acidic (pH< 4) metal salt solutions with
low ionic strength and the large buffering capacity of wool would mean that
the change in pH of the wool would be negligible. In addition, these wools
were irradiated with UVB which does not photobleach wool.
3.3.4. The effect of pH
Fabrics of varying pH were prepared using phosphate buffers and were
irradiated with UVB for four hours while wet. The brightness (Y) of wool
increased after acidification (≤ pH 6.3) compared to untreated wool (Table
3.5).
Treatment
pH
before irradiation after irradiation
X Y Z Y-Z X Y Z Y-Z
untreated 61.9 64.9 55.9 9.0 59.3 62.6 49.4 13.2
3.3 65.0 68.3 59.7 8.5 61.7 65.1 49.1 16.0
4.7 64.3 67.5 58.6 8.9 61.9 65.4 51.4 14.0
6.3 63.7 66.8 57.9 9.0 61.2 64.6 51.4 13.2
7.6 62.4 65.5 56.0 9.5 59.2 62.6 49.5 13.0
8.5 62.0 65.1 56.1 9.0 58.4 61.7 48.7 13.0
9.7 60.5 63.5 53.9 9.6 58.4 61.7 49.2 12.5
Table 3.5 CIE Tristimulus values (X, Y and Z) and yellowness (Y-Z) of wool fabric
of varying pH before and after exposure to UVB for four hours while wet.
The brightening effect has previously been attributed to the destruction of
chromophores within the fibre or the extraction of metal impurities [193].
However, this study has shown that the latter suggestion is unlikely because
levels of copper and iron in acidified wool (pH 3.3) were similar to untreated
fabric (Table 3.1). Wool that was treated with a buffer at pH 7.6 was possibly
contaminated with iron from an impure phosphate salt that was used for the
preparation of the buffer solutions yet the iron did not influence either the
CHAPTER 3
53
initial brightness or the yellowness. Although levels of manganese and zinc
were lower in acidified wool, these metals do not have a strong effect on wool
colour (Table 3.3).
Acidified wool remained brighter after exposure to UVB for four hours while
wet, although the rate of yellowing was greater compared to untreated and
alkali-treated wool. A previous study that acidified wool with a citrate buffer,
and used a phosphate buffer to produce alkaline wool [194], did not find
differences in the rate of yellowing of wool. However, the use of different
buffer systems may account for this result.
The production of HTA after photo-irradiation is less in acidified wool (Figure
3.6) compared to untreated and more alkaline wool. Although the production of
HTA from reaction with TA and hydroxyl radicals requires a mildly alkaline
medium, it is unlikely that the lower production of HTA in acidified wool is
due to changes in pH. The contribution of the fabric to the pH of the TA
solution is negligible when the TA is prepared in an excess of phosphate buffer
(pH 7.6). Also, the production of HTA was reduced in wool that was adjusted
to a pH of approximately 7.6, which was the same pH as the buffer. While
acidification did not extract iron, other species that enhance hydroxyl radical
production may have been removed, or alternatively, species that can quench
hydroxyl radicals could have been generated. Interestingly, hydroxyl radical
production after exposure to a UV light source in these wools is negatively
correlated to the change in yellowness (linear regression, R2 = 0.48).
CHAPTER 3
54
Figure 3.6 Corrected fluorescence intensity after irradiation with UVA for one hour
of HTA (ex= 315 nm, em= 425 nm) of solutions in contact with fabric adjusted to pH
3.3, 4.7, 6.3, 8.5 and 9.7.
3.3.5. Carbonyl production in photo-irradiated wool
The assay of carbonyls in soluble proteins is relatively easy compared to
fibrous proteins such as wool, which require solubilising by hydrolysis. Wool
requires anaerobic digestion to prevent the formation of additional carbonyls
during hydrolysis and it possible that some protein oxidation may have
occurred during this process despite efforts to eliminate oxygen. In addition,
proteins can yield carbonyls as a result of amino acid decomposition during
digestion [50]. Since the samples were digested using the same method, it is
reasonable to assume that similar levels of carbonyls were introduced to each
sample.
It was probable that the increase in hydroxyl radical production in iron-doped
wool would correspond to an increase in the extent of oxidation and carbonyl
production. However, the presence of metals had no effect on carbonyl
production in either unirradiated wool or wet wool exposed to UVB for four
hours (Table 3.6).
0
1
2
3
4
5
6
untreated pH 3.3 pH 4.7 pH 6.3 pH 7.6 pH 8.5 pH 9.7
Flu
ore
sc
en
ce
In
ten
sit
y
`
CHAPTER 3
55
mole carbonyl/gm wool
before
irradiation
after
irradiation
undoped 25 44
Cu 1 26 41
Cu 5 31 41
Fe 1 24 41
Fe 5 25 43
Mn 1 24 44
Mn 5 25 43
Zn 1 23 39
Zn 5 24 38
Table 3.6 Carbonyl content of untreated and copper- (Cu 1, Cu 5), iron- (Fe 1, Fe 2),
manganese- (Mn 1, Mn 2) and zinc- (Zn 1, Zn 2) doped wool before and after
exposure to UVB for four hours while wet.
In biological systems, carbonyl production as a result of metal-catalysed
oxidation is restricted to amino acids on the protein in close proximity to the
metal binding sites. Hydroxyl radicals which cause oxidation are highly
reactive and interact with other species close to where they are generated. In
wool, carbonyl production mediated by metal catalysis may also be limited to
sites around the protein-bound copper and iron. The number of carbonyls
produced by this mechanism may be negligible compared to carbonyls
produced by an alternative oxidation pathway that is independent of metal
content.
3.3.6. Lipid-depleted wool
The concentration of HTA produced in photo-irradiated, lipid-depleted wool
increased compared to untreated wool, and almost to the same extent as iron-
doped wool (Figure 3.7). The level of metals was unaffected by the lipid
extraction process (Table 3.1). This observation implies that internal lipids can
quench hydroxyl radicals, probably by the formation of lipid radicals that can
react with oxygen to produce lipid peroxy radicals (Scheme 3).
CHAPTER 3
56
Figure 3.7 The production of hydroxyl radicals in photo-irradiated untreated, lipid
depleted and iron doped wool.
Scheme 3 Mechanism for lipid quenching of hydroxyl radicals and the production of
lipid peroxy radicals [100].
Unsaturated lipids undergo oxidation and form hydroperoxides more rapidly
than saturated lipids [100] but they are present in low amounts in wool [195].
Another possibility is that a protective species was removed with the lipids. For
example, non-ionic detergents, which are residues from the scouring process,
are associated with the internal lipids in wool (~20% on the weight of extracted
lipids)[196] and their extraction may have an effect. Although hydroxyl radical
production increased in photo-irradiated lipid-depleted wool, removal of lipids
had no effect on the initial colour and the yellowness after exposure to UVB
for four hours (Y-Z 13.5) was similar to untreated wool (Y-Z 13.4).
0
2
4
6
8
10
12
no fabric untreated Fe 3
Flu
ore
sc
en
ce
In
ten
sit
y
Lipid-H + OH Lipid + H2O
Lipid + O2 Lipid-O-O
Lipid-H + OH Lipid + H2O
Lipid + O2 Lipid-O-O
lipid depleted
CHAPTER 3
57
3.3.7. Fibre strength of photo-irradiated wool
Although metal-catalysed oxidation in vivo has little effect on bond cleavage
[81], the presence of iron (~ 50 mg/kg) and possibly copper in dry wool
decreased fibre strength after exposure to a MBTF light source (Table 3.7). The
loss of fibre strength is due to the cleavage of disulfide linkages, and to a lesser
extent, peptide chains [197]. It is possible that at low levels, copper and iron
are associated with the mechanisms of photo-induced rupture of structural
proteins in dry wool. High levels of these metals have been shown previously
to increase embrittlement and the tensile strength of wool after photo-
irradiation due to the formation of protein chain cross-links [198, 199].
In this present study, an increase in burst strength was only observed in copper-
doped fabric that was irradiated wet. An increase in fibre strength is a result of
intermolecular cross-linking and is more likely to occur in wet irradiated wool
when species are labile. The presence of iron in wet-irradiated wool reduced
the wet burst strength.
Sample
number
Wet burst Strength (kPa)
exposed dry exposed wet
untreated 132 (0.73) 141 (0.46)
Cu 2 118 (7.85) 169 (0.61)
Fe 2 104 (7.79) 131 (0.19)
Table 3.7 Mean and standard errors of four wet burst strength measurements
(kPa) of untreated and copper- and iron-doped wool after exposure to a MBTF lamp
for four weeks.
3.3.8. Possible sources of adventitious metal contamination of
processed wool
This study has shown that soaking wool in dilute solutions of iron and copper
(2.5 ppm) at ambient temperature and for a relatively short period of time is
sufficient to increase the level of these metals in wool to the extent that its
colour and photostability is affected. Levels of 0.3 ppm of copper and 0.6 ppm
of iron have been detected in certain water supplies [200] and at this level, iron
can discolour wool when the temperature of the solution is elevated [192].
CHAPTER 3
58
Therefore it is possible that undyed wool could be contaminated with metals
during processing and laundering and affect its colour.
The Sheep CRC has processed a bale of white wool to measure its colour at
each stage through processing [201]. The trace metal content of wool fibre was
also determined at varying stages of the processing pipeline. The commercially
dyed fibre was not available for analysis in this present study. The dyeing
procedure for wool requires prolonged treatment at elevated temperatures and
is usually carried out in stainless steel vats. This process is a possible source of
contamination from trace metals, however wool is usually dyed under acid
conditions and this should limit the uptake of metal ions [6] .
With the exception of zinc, scouring effectively reduced the metal content of
greasy wool. The combing and carding processes, which remove surface
contaminants from the fibre, had little additional effect. The shrink-resist
process involved a metal catalysed chlorine-free / hydrogen peroxide treatment.
The copper, calcium and magnesium content of the shrink-proofed wool
increased whereas the level of zinc was reduced. The high level of copper in
the shrink-resist wool suggests that this metal is possibly the catalyst for the
treatment. The level of 25 mg/kg of copper in the shrink-resist fibre potentially
could increase the photosensitivity of wool.
Treatment Ca Cu Fe Mg Mn Zn
greasy 495 48 45 67 6.5 138
scoured 56 5 29 3.5 0.7 123
combed 26 4 13 2.5 0.3 108
carded 32 11 20 3.5 0.4 110
shrink-resist 244 25 13 27 0.5 62
Table 3.8 Trace metal (mg/kg) content of wool fibre as greasy wool and after
scouring, combing, carding and a shrink-resist treatment.
CHAPTER 3
59
3.4. SUMMARY
Of the transition elements examined in this study, iron was the only metal to
enhance the production of hydroxyl radicals in photo-irradiated wool, even at
relatively low levels (40 mg/kg). In contrast manganese acted as an
antioxidant, possibly by preventing the production of hydroxyl radicals, either
by reacting with superoxide or hydrogen peroxide or by inhibiting the catalytic
action of iron. However, doping with manganese did not afford any protection
to wool and manganese-doped wool photoyellowed to the same extent as iron-
doped wool. The presence of copper did not affect hydroxyl radical production
yet the rate of photoyellowing of copper-doped wool was greater than iron- and
manganese-doped wool. Photoyellowing in wool may not be a direct result of
hydroxyl radical production catalysed by metals. Alternatively, hydroxyl
radical production in copper- and iron-doped wool may be similar, and
hydroxyl radicals that are produced in copper-doped wool may react with
amino acids on the protein in preference to terephthalic acid.
For wools of varying pH, the negative correlation between hydroxyl production
with the change in yellowness after exposure to UV light suggests that
hydroxyl radicals are not the primary cause of photoyellowing in these wools.
The presence of ascorbic acid, citric acid and EDTA increased the rate of
hydroxyl radical production in photo-irradiated wool whereas desferal and
DTPA effectively reduced production. While there is no apparent correlation
between yellowing and the production of hydroxyl radicals, it would be
interesting to determine if the presence of these reagents influences the fibre
strength and the rate of yellowing in wool exposed to UV.
If metals such as copper and iron are involved in the mechanisms of protein
oxidation, then carbonyl production could increase in the presence of these
metals. Although this effect was not observed in photo-irradiated wool, it
remains possible that carbonyls are produced by metal-catalysed oxidation but
to a lesser degree than via other oxidative mechanisms.
CHAPTER 3
60
Compared to untreated wool, wool doped with iron and copper has been shown
to induce a colour change and loss of fibre strength after exposure to UV. It is
therefore probable that copper and iron within the fibres could have some
influence on the colour and strength of contaminated wool and fleece wools.
However, the integration sites of metals in doped wool may be different than in
fleece wool.
CHAPTER 4
61
CHAPTER 4. TRACE METAL CONTENT AND COLOUR OF FLEECE WOOL
4.1. INTRODUCTION
Trace metals are always found in natural protein fibres (Table 4.1) and the
content is a result of the combined effect of genetic and nutritional differences
and exposure to the environment. Trace metals are associated with the colour
of fleece wool by acting as mordants for natural pigments that stain wool [108]
or as constituents of dust that can occlude in the fibre and affect the colour of
scoured wool [107, 202]. In this study, copper and iron have been shown to
influence the change in colour, fibre strength and hydroxyl radical production
in metal-doped wool, even at relatively low levels. Therefore it is reasonable to
assume that these elements may have a similar influence on fleece wools and
affect their colour when exposed to the environment and solar radiation.
Trace metal content
Ca Cr Cu Fe Mg Mn S Zn
wool 125 0.3 7.6 9 15 0.2 35833 124
royal spoon bill feathers 570 0.3 7 43 133 19 32400 111
silk fabric 1515 0.2 2.1 8 51 1.65 1061 5.55
angora rabbit fur 230 0.2 12 8 35 0.2 40300 182
mink fur 96 0.35 8.6 7 13 <0.1 54450 171
Table 4.1 Trace metal content (mg/kg) of cleaned protein fibres determined using the
method described in Chapter 2.
All animal species require iron, copper, selenium and zinc for the maintenance
of the immune system and health [203], particularly zinc for sheep [204].
Copper deficiency has also been shown to decrease resistance to infection
[205]. The status of the immune system and responses to pathogens can affect
the colour and yellowing of wool on sheep. An example is fleece rot and
“canary” yellowing, which is caused when the fleece is exposed to periods of
prolonged wetting, and results in an increase in the population of Pseudomonas
aeruginosa [206], the predominant bacterium present in the fleece [206]. These
CHAPTER 4
62
bacteria exude phenazine derivatives which are pigments that discolour wool
[21]. Resistant animals have a higher antibody response to Pseudomonas
aeruginosa [207] and have wool with higher zinc and lower copper content
[13] than susceptible sheep. Resistance to fleece rot is also associated with
white and bright greasy wool [208] and is genetically correlated to wool colour
[209]. Like fleece rot, resistance to flystrike by Lucilia cuprina is influenced by
greasy wool colour [210] and the immune system which affects the severity of
skin lesions caused by the larvae [211]. Wound exudates stain wool and are
also a source of trace metals [117] that can be adsorbed by wool fibres.
Trace metals have an important role in the function and metabolism of genetic
molecules and the transfer of genetic information [212]. The trace metal status
and metabolism is controlled by genetics as a response to endocrine, nutritional
and environmental stimuli. Sheep can be selected for low and high copper
status [205] and susceptibility to copper toxicoses is dependent on sheep breed
[213]. Biopsies have shown that the metal content of sheep livers is heritable
and the h2 estimates for copper, zinc and selenium are 0.60 ± 0.32, 0.52 ± 0.28
and 0.43 ± 0.29 respectively [214]. The level of zinc and copper in wool is
dependent upon breed [215]. For example, wool from white Egyptian Merinos
has less iron, manganese and copper than other breeds of Egyptian sheep [151]
.
The relationship between trace metals and wool colour is complex. To further
examine the relationship, this study determined the range and variability of the
trace metal content and yellowness of 700 wool samples collected from 419
sheep in the Sheep CRC IN flocks that were located at eight sites throughout
Australia. The copper and iron content has a significant effect on the colour
and photostability of metal-doped wool fabric and this study determined
whether their influence is similar in fleece wools. Differences due to ageing
were evaluated by comparing wool collected from one-year old sheep and the
following year as two year olds. This enquiry expanded on previous studies of
trace metals in wool, which were limited to sampling a maximum of 100 sheep
[13, 108, 120, 134, 216].
CHAPTER 4
63
4.2. MATERIALS AND METHODS
4.2.1. Selection, preparation and analysis of samples
Wool was sampled from sheep in the Sheep CRC Information Nucleus flocks
which are located at sites in Hamilton and Rutherglen (Victoria), Turretfield
and Struan (South Australia), Cowra and Kirby (New South Wales) and
Katanning (Western Australia). The samples were collected by removing
approximately 100 grams of wool from a 250 x 250 mm area on the mid-side
of each Merino sheep. Collecting wool from the mid-side area provides
consistency in sampling because different parts of the body yellow to different
extents [217]. A portion of each greasy wool sample was archived by the Sheep
CRC and the remaining fibre was scoured and fibre properties, including
colour (IWTO-56-03) and mean fibre diameter (Optical Fibre Diameter
Analyser), were measured by the Australian Wool Testing Authority (AWTA).
The average yellowness (Y-Z) for the three years of sampling ranged from 8.1
to 8.3 with a minimum of 5.6 and a maximum of 22.8 (Table 4.2). The
distribution of yellowness values for the 2009 samples shows that the majority
of samples have Y-Z values between 7 and 9 (Figure 4.1).
Year of sampling
2008 2009 2010
Mean 8.3 8.1 8.3
Standard Deviation 0.75 0.87 1.16
Sample Variance 0.56 0.76 1.33
Range 8.8 10.4 16.9
Minimum 6.3 5.6 5.9
Maximum 15.1 16.0 22.8
Confidence Level(95.0%) 0.04 0.03 0.05
Table 4.2 Descriptive statistics of wool yellowness (Y-Z) (IWTO 2003 test method)
of all Merino sheep in the Sheep CRC IN Flock
CHAPTER 4
64
Figure 4.1 Distribution of yellowness measurements (Y-Z) of samples collected in
2009 from Merino sheep in the Sheep CRC IN Flock.
In 2008, a total of 1289 wool samples were available for analysis from one
year old Merino sheep in the IN flock that were born in 2007. The following
year in 2009, a total of 2552 samples were collected from the surviving sheep
from the 2007 drop and those born in 2008. The available samples in 2010
were from the survivors of the 2008 drop and sheep that were born in 2009
(Table 4.3). It was not feasible to prepare and analyse all of these samples for
trace metal content.
Only wool samples with the extremes of yellowness were of interest and were
selected for analysis. These samples included the 10 most yellow and 10 least
yellow wools from one year-old sheep that were located at each of the IN sites
and collected in 2008 and 2009. Wool that was taken from the surviving sheep
in 2009 and 2010 as two year-olds was analysed for trace metal content to
determine variations associated with ageing. A total of 281 sheep were sampled
as one year olds and two year olds. Because of severe drought, there were no
lambs born in 2007 at the Trangie site and consequently there were no
available samples from this site in 2008. Seven highly yellowed samples
collected in 2010 from the 2009 drop were included in this study as well as the
entire collection of 122 samples from Hamilton in 2008 that were analysed to
0
50
100
150
200
250
300
350
5.6 6.8 8 9.2 10.4 11.6 12.8 14 15.2
Fre
qu
en
cy
Yellowness (Y-Z)
CHAPTER 4
65
estimate the distribution and range of trace metal content in one flock. The
sampling protocol is tabulated (Table 4.3).
Year Number of available samples
Number of samples analysed for
trace metal content
2007 drop 2008 drop 2009 drop 2007 drop 2008 drop 2009 drop
2008 1289 242
2009 916 1636 175 170
2010 1168 1304 107 7
Table 4.3 Number of available samples from the IN Flock and the number of samples
analysed for trace metal content.
Sub-samples of approximately 0.55 gm were taken by randomly selecting fibre
from several areas of a 20 gm sample that was scoured and blended wool at
AWTA. The test specimens were prepared and analysed for quantitative
determination of bound trace metals using the protocol described in Chapter 2.
As a quality control measure, random samples were analysed in duplicate for
each batch of approximately 20 single analyses and a control sample was
included for each batch of approximately 50 samples.
Elemental analysis and colour data are stored in a database maintained by the
Co-operative Research Centre for Sheep Industry Innovation.
A Merino ram’s fleece used for the development of the method for trace metal
analysis, and wool from a shedded and coated Merino sheep (Sharlea), were
used to compare the trace metal content of the base, mid-section and the tip of
the fibre.
Eight greasy mid-side wool samples were sourced from the 2008 collection of
samples and were selected based on extremes of copper (3.8-15 mg/kg) and
iron content (8.8-58.8 mg/kg). Both whole fibre and the section of fibre 20 mm
from the root were scoured using the protocol for maximising whiteness. The
trace metal content was determined and the yellowness was measured using the
method discussed in Chapter 5.
CHAPTER 4
66
4.2.2. Statistical analysis
Although two samples were taken from the same animal over successive years,
annual variation in climate, diet, husbandry practices, as well as the possible
effect of age, mean that colour and trace metal content can vary, therefore the
wool samples were considered to be independent of one another.
Outliers with unusually high zinc content (> 200 mg/kg, n=3) were removed
and because the distributions were skewed, the data were transformed to log10
values prior to stepwise (Type 1) multiple regression analysis using SPPS-
PASW (Predictive Analytics SoftWare) Version 18 and Pearson correlations
and T-tests assuming unequal variance using Microsoft Excel 2003.
4.3. RESULTS AND DISCUSSION
4.3.1. Distribution and range of trace metal content
Since only approximately 10% of the available samples were selected for this
study, and selection was based on the extremes of colour, the entire collection
of wool samples from one randomly selected flock were analysed to estimate
the likely distribution of trace metals in all samples from the IN Flocks. Wools
sampled from one-year old sheep (n=122) located at Hamilton in 2008 were
compared to the remainder of the samples analysed for trace metal content (n =
575) to determine if selection based on colour could incur an unfavourable bias
when determining correlations and relationships.
The yellowness of wool from the Hamilton flock (Y-Z 6.3-9.5) and wool
selected for extremes of yellowness (Y-Z 5.6-22.8) were normally distributed.
Selection for colour extended the range of values and positively skewed the
distribution (Figure 4.2) similar to the yellowness values of the entire set of
samples collected in 2009 from IN flocks (n = 2552) (Figure 4.1).
CHAPTER 4
67
2008 Hamilton flock Selected for extremes of
yellowness
Mean 7.8
Minimum 6.3
Maximum 9.4
Mean 8.5
Minimum 5.6
Maximum 22.8
Mean 136
Minimum 93
Maximum 180
Mean 111
Minimum 64
Maximum 200*
Mean 8.0
Minimum 5.7
Maximum 10.6
Mean 10.8
Minimum 3.8
Maximum 76.5
Figure 4.2 Distribution of yellowness (Y-Z) and zinc and copper content (mg/kg) of
wool samples collected from the entire Hamilton flock of yearling sheep in 2008
(n=122) and samples selected for extremes of yellowness (n = 575).
* three outliers with zinc contents >200 mg/kg were removed from the dataset
0
4
8
12
16
6.3 6.9 7.5 8.1 8.7 9.3
Yellowness (Y-Z)
Fre
qu
en
cy
0
50
100
5.5 8.5 11.5 14.5 17.5 20.5
Yellowness (Y-Z)
Fre
qu
en
cy
0
4
8
12
16
93 111 129 147 165
Zinc content (mg/kg)
Fre
qu
en
cy
0
25
50
75
65 95 125 155 185
Zinc content (mg/kg)
Fre
qu
en
cy
0
2
4
6
8
10
12
5.6 6.8 8 9.2 10.4
Copper content (mg/kg)
Fre
qu
en
cy
0
50
100
150
4 16 28 40 52 64 76
Copper content (mg/kg)
Fre
qu
en
cy
CHAPTER 4
68
2008 Hamilton flock Selected for extremes of
yellowness
Mean 230
Minimum 45
Maximum 664
Mean 234
Minimum 17
Maximum 1643
Mean 25.7
Minimum 11
Maximum 58.8
Mean 18.9
Minimum 7.6
Maximum 78
Mean 17
Minimum 2.8
Maximum 76.5
Mean 17.5
Minimum 0.4
Maximum 203
Mean 1.3
Minimum 0.1
Maximum 3.1
Mean 2.0
Minimum 0.1
Maximum 16
Figure 4.3 Distribution of calcium, iron, magnesium and manganese content (mg/kg)
of wool samples collected from the entire Hamilton flock (n=122) of one year old
sheep in 2008 and samples selected for extremes of yellowness (n = 575).
0
4
8
12
16
40 160 280 400 520 640
Calcium content (mg/kg)
Fre
qu
en
cy
0
50
100
150
20 300 600 900 1200 1500
Calcium content (mg/kg)
Fre
qu
en
cy
0
4
8
12
16
20
10 22 34 46 58
Iron content (mg/kg)
Fre
qu
en
cy
0
50
100
4 16 28 40 52 64 76
Iron content (mg/kg)
Fre
qu
en
cy
0
4
8
12
16
20
24
28
3 21 39 57 75
Magnesium content (mg/kg)
Fre
qu
en
cy
0
50
100
150
3 30 60 90 120 150 180
Magnesium content (mg/kg)
Fre
qu
en
cy
0
4
8
12
16
0.1 0.7 1.3 1.9 2.5 3.1
Manganese content (mg/kg)
Fre
qu
en
cy
0
50
100
150
0.1 3 6 9 12 15
Manganese content (mg/kg)
Fre
qu
en
cy
CHAPTER 4
69
The distribution of copper content of wools selected for colour (3.8-76.5
mg/kg) was also skewed compared to wool from the Hamilton flock (5.7-10.6
mg/kg) whereas the distribution of zinc content appears to be independent of
the sample selection method. The range of zinc in wool from Hamilton (93-180
mg/kg) was similar to the remaining 578 samples (64-204 mg/kg) that were
selected for trace metal analysis. The range of levels of elements in wool
samples from the Hamilton flock were calcium (45-664 mg/kg), iron (11-58.8
mg/kg), magnesium (2.8-76.5 mg/kg) and manganese (0.1-3.1 mg/kg). These
ranges were less than those observed in wools selected based on their colour
which were; calcium (17-1643 mg/kg), iron 7.6-78 mg/kg), magnesium 0.4-
203 mg/kg, manganese 0.1-3.1 mg/kg). The distribution of these elements was
similar in both sample sets.
T-Test analysis assuming unequal variances indicates that there is little
evidence that the means of calcium (p ≤ 0.65) and sulphur (p ≤ 0.58) content of
the Hamilton samples were different from the means of samples selected for
colour. However, the means of copper, iron, manganese and zinc (p < 0.01)
were different for the two samples sets. Although there are statistical
differences, it cannot be assumed that selection based on colour will affect the
evaluation of relationships because the range of metal contents of wool from
the Hamilton flock is within the range in the samples selected based on colour.
4.3.2. The relationship between trace metal content and wool colour
The scatterplot of the colour values X, Y, Z and Y-Z and trace metal content
(n= 697) (Figure 4.4) shows that the relationship between individual tristimulus
values and Y-Z with copper and iron content is not linear. Although yellower
wools tended to have lower levels of these metals, whiter wools can have a
range of levels. The relationship between Y-Z with copper and iron is also
shown in greater detail in Figure 4.5. These results agree with studies that
found higher levels of copper in wools that appeared whiter [122] and less
prone to “canary” yellowing [218] and conflict with the conclusion that sheep
were more likely to produce whiter wool when grazed on copper-deficient soils
inferring that whiter wool had lower levels of copper [11].
CHAPTER 4
70
If the primary determinant of the colour of fleece wools is due to photo-
sensitisation or photoyellowing catalysed by iron and copper, then one would
expect a positive correlation between yellowness and these elements.
Compared to metal-doped wool, the average iron (18.9 mg/kg) and copper
(10.8 mg/kg) content of fleece wools is low. Only 2.7% of samples had copper
contents >22 mg/kg and 3.7% had iron contents >42 mg/kg which were the
minimum levels in doped fabric. Also, the correlation of these metals with Y-Z
is poor suggesting that photo-sensitisation or photo-initiated oxidation
catalysed by copper and iron is not the predominant cause of yellowing in
fleece wools.
Figure 4.4 Scatterplot of the relationship between calcium, copper, iron, magnesium,
sulphur, zinc content with yellowness (Y-Z) and the X, Y, Z tristimulus values.
Ca Cu Fe Mg Mn S Zn X Y Z Y-Z
Ca
Cu
Fe
Mg
Mn
Zn
S
X
Y
Z
Y-Z
CHAPTER 4
71
Multiple linear regression analysis using the stepwise (Type 1) option indicates
that of all the trace metals analysed, magnesium (R2= 0.19) is the strongest
predictor of Y-Z. The relationship was independent of the order of the input of
the variables. Mean fibre diameter, which is correlated to wool yellowness
[219], was also included in the dataset, however this parameter was not
important in the regression model. The positive linear relationship between Y-
Z with calcium, magnesium and manganese is shown in Figure 4.4 and in
greater detail in Figure 4.5. Pearson correlation coefficients confirm that
overall there is a strong positive linear relationship between Y-Z and calcium
(0.39), magnesium (0.43) and manganese (0.41) content of wool. However the
correlation is only important in wool with higher Y-Z values (Table 4.4).
Calcium and magnesium contents are also strongly positively correlated with
one another (0.90) and are positively correlated to manganese (calcium, 0.58
and magnesium, 0.65). Previous studies have shown that there is a strong
relationship between the calcium and magnesium status of sheep [220] and that
wool content is positively correlated with the level of these elements in serum
[221]. The association between calcium and magnesium has also been
observed previously in transverse profiles of human hair [222].
Y-Z 5.6 - 7 Y-Z 7.1- 8.4 Y-Z ≥ 8.5 All wool
n = 93 n = 358 n = 246 n = 697
Calcium -0.23 0.10 0.50 0.39
Copper 0.10 -0.09 -0.12 -0.08
Iron -0.34 -0.10 -0.02 -0.34
Magnesium -0.12 0.14 0.54 0.43
Manganese -0.12 0.08 0.62 0.41
Zinc 0.04 -0.20 -0.17 -0.39
Table 4.4 Pearson correlation coefficients of yellowness (Y-Z) with calcium, copper,
iron, magnesium, manganese and zinc content of white, average and yellower wools.
Multiple regression analyses exclude parameters that are strongly correlated to
the primary determinant and therefore calcium and manganese were not
included in the model. As a result, zinc is of secondary importance in multiple
regression models and combined with magnesium is associated with 39% of
the variability in Y-Z. Overall, zinc content is negatively correlated to wool
CHAPTER 4
72
yellowness, although this is dependent on the initial yellowness (Table 4.4) and
the relationship is not linear (Figure 4.4, Figure 4.5). However it is not possible
to draw definitive conclusions from this data because wools from at least two
IN flock sites were contaminated with zinc sulphate which is a common
treatment for footrot in sheep. This practice was only apparent when some
wool samples (n = 3) were found with levels of zinc greater than 200 mg/kg
with a maximum of 2060 mg/kg. In these cases the particular sheep most
probably fell into the treatment bath containing concentrated zinc ions.
Although these three samples were excluded from the dataset, the contribution
of zinc treatments to the content of the remaining wool samples is uncertain.
Figure 4.5 The relationship between yellowness (Y-Z) with iron, copper, calcium,
magnesium, manganese and zinc content (mg/kg).
R² = 0.064
5
10
15
20
25
0 20 40 60 80
Ye
llo
wn
es
s (Y
-Z)
Iron content (mg/kg)
R² = 0.0012
5
10
15
20
25
0 10 20 30 40
Ye
llo
wn
es
s (Y
-Z)
Copper content (mg/kg)
R² = 0.381
5
10
15
20
25
0 500 1000 1500
Ye
llo
wn
es
s (Y
-Z)
Calcium content (mg/kg)
R² = 0.412
5
10
15
20
25
0 50 100 150 200
Ye
llo
wn
es
s (Y
-Z)
Magnesium content (mg/kg)
R² = 0.415
5
10
15
20
25
0 3 6 9 12 15 18
Ye
llo
wn
es
s (Y
-Z)
Manganese content (mg/kg)
R² = 0.122
5
10
15
20
25
60 90 120 150 180 210
Ye
llo
wn
es
s (Y
-Z)
Zinc content (mg/kg)
CHAPTER 4
73
Brightness (Y) is a more appropriate descriptor of differences in the colour of
metal-doped fabric, which can appear discoloured whilst having similar Y-Z
values to untreated wool. Therefore the correlations with Y and metal content
were determined for the fleece wools used in this study (Table 4.5). With the
exception of zinc, all metal contents were negatively correlated to brightness.
The results are similar to metal-doped fabrics which become duller as the iron
and copper content increases (Table 3.3). However the negative correlation
between brightness and manganese content was not observed in doped wool.
Y-Z 5.6 - 7 Y-Z 7.1- 8.4 Y-Z >=8.5 All wool
n = 93 n = 358 n = 246 n = 697
Calcium -0.25 -0.34 -0.43 -0.38
Copper -0.23 -0.41 -0.14 -0.25
Iron -0.45 -0.45 -0.23 -0.29
Magnesium -0.10 -0.25 -0.44 -0.32
Manganese -0.32 -0.08 -0.39 -0.28
Zinc 0.02 -0.15 0.02 0.01
Table 4.5 Pearson correlation coefficients of brightness (Y) with calcium, copper,
iron, magnesium, manganese and zinc content.
4.3.3. Effect of age on trace metal content and wool yellowness
T-test comparisons assuming unequal variance of the Y-Z values of wool
sampled from 281 sheep in the IN flocks at one and two years of age shows
that yellowness does not change with age (p = 0.14). The iron (p= 0.31) and
zinc content (p= 0.32) remain unchanged whereas copper levels increase (p<
0.01). The reduction in calcium, magnesium and manganese with age was
statistically significant (p< 0.01) (Table 4.6) although this was not as apparent
in the whitest wools (Table 4.8).
CHAPTER 4
74
Mean (n = 281) P value
one year old two year old
Calcium 255 174 <0.01
Copper 8.6 12.5 <0.01
Iron 20 19 0.31
Magnesium 20 10 <0.01
Manganese 2.1 1.3 <0.01
Zinc 126 119 0.32
Y-Z 8.3 8.2 0.14
Table 4.6 T-test comparison assuming unequal variance of yellowness (Y-Z) and
calcium, copper, iron, magnesium, magnesium and zinc content of wool sampled from
the same sheep at one and two years of age.
4.3.4. Calcium, magnesium and wool colour
Elemental analysis results of the yellowest (Y-Z >13) and whitest (Y-Z <6),
wools from sheep that were sampled as one and two year-olds were tabulated
to demonstrate that the yellowest wools have higher levels of both calcium and
magnesium compared to white wool (Table 4.8). Interestingly, the ratio of
calcium to magnesium content is lower in wool from one year old sheep,
particularly those which are highly yellowed (Figure 4.6).
yellowest wool
Sheep Genetics ID Age (years) Ca Cu Fe Mg Mn Zn Y-Z
26IN082008080789 1 1250 9 14 170 4.5 105 15.8
2 801 16 12 65 1.7 112 9.3
26IN072007070202 1 1028 5.5 16 120 5.6 85 15.1
2 394 8 17 28 2.1 102 8.5
26IN022008080294 1 845 8 10 74 7.9 125 14.4
2 320 7.7 14 18 5 130 12.6
26IN082007070216 1 833 8.2 13 114 1.5 133 13.8
2 55 11 28 5 0.5 111 8.6
26IN032008080139 1 724 8 14 78 10 81 13.7
2 380 10 13 27 4.7 96 11.9
26IN022008080450 1 1643 10 22 176 10 96 13.6
2 600 7.3 32 48 4.5 110 10.3
26IN072007070138 1 1036 7.2 25 122 6.2 94.5 13.4
2 253 6 12 17 1.5 124 8.5
Table 4.7 Yellowness (Y-Z) and calcium, copper, iron, magnesium, manganese and
zinc content of the yellowest (Y-Z >13) wool from sheep sampled at one and two
years of age.
CHAPTER 4
75
whitest wool
Sheep Genetics ID Age (years) Ca Cu Fe Mg Mn Zn Y-Z
26IN082008080160 1 313 9 28 17 2.3 122 5.6
2 410 11 19 26 1.3 104 7.7
26IN082008080296 1 115 7 28 4 1.1 119 5.7
2 196 18 14 10 0.8 123 8.2
26IN082008080382 1 160 9 67 9 2.4 95 5.8
2 69 16 30 4.5 0.5 92 7.6
26IN082008081209 1 213 9 26 11 1.6 109 5.8
2 200 11 13 10 0.9 117 8
26IN082008080173 1 44 5 22 2 0.4 110 5.9
2 57 10 16 4.3 0.4 105 7.5
26IN082008080362 1 92 11 63 4 1.1 113 5.9
2 81 11 24 3.9 0.6 104 7.9
Table 4.8 Yellowness (Y-Z) and calcium, copper, iron, magnesium, manganese and
zinc content of the whitest wool (Y-Z<6) from sheep sampled at one and two years of
age.
Figure 4.6 The effect of the age of sheep on the relationship between yellowness
(Y-Z) and the calcium and magnesium ratio of wool.
There are several possible sources of calcium and magnesium and reasons for
the age-related differences. Contamination from airborne soil and water could
be a source of calcium and magnesium, which are the most abundant divalent
5
10
15
20
25
0 50 100 150
Ye
llo
wn
es
s (
Y-Z
)
Calcium / Magnesium
one year olds
two year olds
CHAPTER 4
76
cations in fresh water [223] and clay soils [224]. It is unlikely that the level of
metals in wool from soil and water is associated with age because sheep in the
same flock would be exposed to the same environmental contamination
regardless of age, although differences in fleece density may have an effect.
Ewe’s milk is high in calcium (1930 mg/kg) and magnesium (180 mg/kg)
[225]. The calcium status of sheep is highest in milk-fed lambs and declines
after weaning [226] which can be up to 20 weeks of age [227]. Since the
calcium status of sheep is correlated to wool content [221], nutritional
differences between milk and plant fodder could account for differences in
calcium and magnesium content in wool from one year old and two year-old
sheep.
Another likely source of trace metals is suint which is carried passively along
the length of the wool fibre [228]. Soluble metal ions can be readily adsorbed
by wool, particularly oxidised and carbonylated wool [130]. Suint which has
high levels of calcium (7755 mg/kg) and magnesium (95 mg/kg) and iron (600
mg/kg) (Table 4.9) and levels may vary with age.
suint wool wax
Potassium (mg/kg) 187500 6750
Calcium (mg/kg) 7755 270
Iron (mg/kg) 600 700
Magnesium (mg/kg) 95 140
Manganese (mg/kg) 10 10
Copper (mg/kg) 10 10
Zinc (mg/kg) ― ―
Table 4.9 Calcium, iron, magnesium, manganese, copper and zinc content (mg/kg) of
suint and wool wax [229].
Suint composition [13, 230] as well as the microbial population on the fleece
are associated with within and between breed variation in wool colour [231].
They are also associated with fleece rot and wool yellowing in older sheep,
although the correlation in younger animals is poorer [232]. Sheep which
CHAPTER 4
77
produce greater volumes of suint [10] that has higher levels of boron,
manganese and iron and lower levels of sodium, phosphorus and zinc tend to
have yellower wools [231]. In comparison, yellower wools used for this study
had higher manganese and lower zinc contents, confirming an association
between suint with colour and trace metal content. However these wools had
lower levels of iron, which suggests there may be other processes that affect
the iron content of wool.
Non-scourable pigments that cause “canary” yellowing are found in the yolk,
which is a combination of suint, wool wax and bacterial residues [13]. These
pigments are predominantly phenazine derivatives produced by the bacterium
Pseudomonas aeruginosa [21]. Bacteria exude polymeric substances which
form a biofilm that assists cohesion between cells and adhesion to surfaces, and
phenazines influence the structure of the biofilm [233].
Calcium and magnesium are required for the production and adhesion of
biofilms [234] and when over the threshold concentration [235] these cations
can change the electrostatic charge of a substrate that attracts bacteria. They are
also required for the formation of covalently bonded linkages with bacteria and
the substrate [236]. Therefore, wools with high levels of both calcium and
magnesium are suitable substrates for the proliferation of Pseudomonas
aeruginosa and other bacteria. Higher magnesium and calcium levels and
lower levels of zinc have been found previously in greasy wools which have a
propensity to yellow in warm humid conditions that encourage bacterial growth
[13]. These conditions do not affect scoured wool to the same extent as greasy
wool [237] because bacteria, suint and metal contaminants are removed from
the fibre.
Pseudomonas aeruginosa also require iron for cell growth which is initiated
when there is sufficient intracellular iron [238]. They acquire iron and other
metals including copper, zinc and manganese [239] by producing and secreting
siderophores, which are small organic compounds that can scavenge metals
from minerals by forming soluble metal chelates which can be readily adsorbed
[240].
CHAPTER 4
78
Although it cannot be confirmed without further experiments, it is probable
that the highly yellowed wools analysed in this study are discoloured by
pigment secreting bacteria such as Pseudomonas aeruginosa. The yellowest
wools (Y-Z >13) have high calcium and magnesium, possibly from residues of
biofilm formation and low iron, copper and zinc and contents (Figure 4.4)
suggesting that depletion could be caused by sequestration by bacteria. Further
study is also required to determine if wools with high calcium and magnesium
content are more prone to bacterial colonisation on the fibre surface or whether
high levels of these elements in wool are a result of accumulation by
Pseudomonas aeruginosa. These highly yellowed wools were from young
sheep which are more likely to have an immature immune response to this
bacterium [207] and are more susceptible to fleece rot compared to older sheep
[20]. Younger sheep also produce less wax, which is known to provide a
barrier to moisture and bacterial infection [241].
If pigments secreted by bacteria contribute significantly to the discoloration of
fleece wools during growth, it may be feasible to inhibit bacterial growth and
the associated yellowing by reducing the level of metals within wool, and the
suint and wax that cover fibres. Ethylene diaminetetraacetic acid (EDTA)
chelates metals and disrupts bacterial biofilms and is more effective at killing
Pseudomonas aeruginosa than the antibiotic gentamycin, although a combined
treatment of antibiotics and EDTA increases the efficacy and completely kills
biofilm cells [242]. Chelating agents can be applied off shears and have been
used to remove copper from fleeces, although these compounds discoloured the
wool [134]. Also, antimicrobial agents may be applied as a backline treatment
to remove skin bacteria in order to improve wool colour [231]. Therefore an
application of a non-toxic chelating agent that does not stain wool, together
with a suitable antibiotic, may prevent yellowing and fleece rot caused by
Pseudomonas aeruginosa. Alternatively, improvements could be made by
selecting sheep with a higher antibody response to Pseudomonas aeruginosa
[207] and which produce more protective wool wax, denser fleeces [241] and
less alkaline suint [231] that will reduce the availability and uptake of essential
metals by bacteria [239].
CHAPTER 4
79
4.3.5. Limitations of this study
4.3.5.1. Effect of environment
It was anticipated that regional effects on the trace metal content of wool could
be evaluated using wool which was collected from sheep raised in different
sites across Australia. Although site differences were observed, inconsistencies
in sampling protocol and sampling times meant that time-independent
evaluation between IN sites was not possible. Tip shearing to remove foetal
wool prior to wool sampling is a common practice when conducting
experiments on sheep. Sheep at Struan and Katanning were tip shorn at
weaning and sampled at 15-16 months with 11-12 months of wool growth,
whereas sheep from Rutherglen and Cowra were also tip shorn but sampled as
yearlings with 6-7 months of wool growth. Sheep from Hamilton, Turretfield
and Kirby flocks were not tip shorn and samples collected from these sheep
represented ten to eleven months of wool growth.
Although tip shearing was not practiced in 2009 and 2010, the wool samples
did not represent the same period of wool growth. For example, sheep at
Katanning that were born in 2007 were sampled with 10−12 months of wool
growth whereas the 2008 drop sheep were sampled with 15 months of wool.
The 2007 drop sheep at Cowra were sampled with almost 13 months of wool
growth whereas the 2008 drop sheep were sampled at 10 months. The effect of
the time of exposure to the environment on the level of trace metals in wool is
likely to be important and it is impossible to correct for these differences in
wool growth across sites. Equally, evaluation of the genetic and environmental
effects on wool colour is likely to be less accurate using samples with different
amounts of wool growth.
4.3.5.2. Analysing different regions along the wool staple
The variation in the trace metal content along the length of wool fibre taken
from coated and shedded sheep is low, whereas the tips of wool from pasture-
raised sheep have higher levels of calcium, iron, magnesium and manganese
than the remainder the fibre (Table 4.10). The tips receive the maximum
exposure to dust [108] and sunlight, both of which can increase the trace metal
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80
content and yellowness of wool [134], and the extent of penetration will vary
between fleeces.
Ca Cu Fe Mg Mn S Zn
Pasture raised
sheep
root 232 3.8 9.6 52 0.2 34450 133
mid 180 4.2 8.9 42 0.2 34650 124
tip 588 4.2 16.5 138 4.6 32200 138
Shedded and
coated sheep
root 248 12 13 30 <0.2 34000 148
mid 96 17 9 13 <0.2 33950 141
tip 194 27 10.5 24 0.2 34600 153
Table 4.10 Comparison of the trace metal content (mg/kg) and yellowness (Y-Z) of
the tip, mid-section and base of wool fibre from pasture-raised sheep and coated and
shedded sheep (Sharlea).
The trace metal content of whole fibre compared to the root section of scoured
wool shows that the contribution of the tips varies between sheep (Table 4.11)
and is more apparent for the calcium, magnesium and iron contents. Including
the tips had little effect on the trace metal content of samples 2, 4, 5, 6 and 8
whereas the difference between whole fibre and tip free fibre was greater for
samples 1, 3 and 7.
Sample
Number Fibre Ca Cu Fe Mg Mn Zn
1 tip free 69 5.9 8.1 8.5 0.3 99
whole 215 8.4 59 32 1.9 133
2 tip free 99 4.1 6.5 10 0.3 89
whole 43 3.8 8.8 2.2 0.8 84
3 tip free 72 6.1 6.2 6.8 0.2 113
whole 534 11 36 48 2.9 89
4 tip free 109 6 6.4 5.6 0.2 105
whole 174 5.9 9.1 10 0.8 93
5 tip free 78 3.3 7 10 0.2 128
whole 52 4.5 22 5.9 0.7 126
6 tip free 56 4.1 7.1 6.2 0.2 123
whole 52 5.6 8.9 3.8 0.1 98
7 tip free 50 5.1 8.6 4.8 0.2 124
whole 484 15 20 35 0.7 124
8 tip free 249 5 5.7 22 0.4 91
whole 311 5.9 7.6 22 0.6 99
Table 4.11 Comparison of the trace metal content (mg/kg) of scoured whole fibre
and the root section of scoured wool.
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81
Including the tips can also increase Y-Z values by between 0.2 and 1.6 Y-Z
units (Figure 4.7) and the variation is likely to be greater when sampling a
larger number of sheep.
Figure 4.7 Comparison of the yellowness of scoured whole fibre and the root section
of scoured wool.
It is not possible to accurately assess the contribution of each biochemical and
environmental process to the overall trace metal content and yellowness when
analysing whole fibre from pasture-raised sheep. By analysing the base section
of the fibre, or wool from shedded and coated sheep, the effect of dust and
environmental exposure is minimised and the level of trace metals and colour
of the wool is mainly influenced by genetics and nutrition. However, trace
elemental analysis of whole fibres from pasture-raised sheep could identify
animals which may be more resistant to yellowing and weathering from
exposure to the environment.
4.4. SUMMARY
If the predominant influence on the colour of fleece wools is photoyellowing
catalysed by iron and copper, then one would expect the relationship between
0
1
2
3
4
5
6
7
1 2 3 4 5 6 7 8
Ye
llo
wn
es
s (
Y-Z
)
Sample number
tip free fibre whole fibre
CHAPTER 4
82
colour and metal content to be similar to metal-doped photo-irradiated wool.
The relationship of the brightness (Y) of 700 fleece wools with the iron and
copper content was similar to doped wools in this study, although the levels of
copper and iron in the majority of fleece wools were lower than the doped
wools. Unlike doped wool, the yellowness of the fleece wools was not
positively correlated to the copper content and the yellowest wool had low
levels of copper. Although fleece wools with low yellowness values had higher
levels of iron, similar to doped wool, yellower wool had lower levels of iron
and the correlation between yellowness and iron content was poor. It is
therefore probable that the intrinsic levels of copper and iron in fleece wools
are generally too low to have an effect and that photoyellowing catalysed by
iron and copper is not a major influence on yellowness.
In contrast, calcium, magnesium and manganese were positively correlated to
yellowness although this effect is influenced by heavily yellowed wools. This
result is consistent with the hypothesis that yellowing is a result of staining by
exudates from bacteria which require calcium and magnesium to form stable
biofilms. Bacteria also sequester metals and there is evidence that the heavily
yellowed wools are depleted of copper, iron and zinc. The explanation for a
positive relationship between manganese and wool yellowness is unknown.
This study has shown that the processes that influence wool colour are
numerous and complex, that identifying the primary determinants is difficult
and that the relationships between colour and trace metal content depend on the
yellowness value. The data generated from this study is currently being
evaluated by the Sheep CRC to determine if the trace metal content of wool is
heritable. Fleece density, wax production, which affect the structure of the
fleece and suint composition are heritable traits which could influence the level
of exogenous metals in wool. The trace metal status in the serum of sheep is to
some extent controlled by genetics and is associated with the content of the
endogenous metals in wool. It is therefore probable that the trace metal content
of wool is heritable.
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83
CHAPTER 5. A METHOD TO MEASURE THE COLOUR AND PHOTOSTABILITY OF SMALL FLEECE WOOL SAMPLES
5.1. INTRODUCTION
In the three years of sampling sheep in the IN flock, approximately 70% of the
sheep had wool with a yellowness value within the acceptable limit (Y-Z< 8.5)
and the incidence of highly yellowed wool averaged 1.3% (Table 5.1). Sheep
in the IN flock are from a diverse range of bloodlines of Australian sheep and
the proportion of sheep that have undesirable wool colour is likely to be similar
to the entire Australian Merino sheep population.
Year of sampling
2008 2009 2010 Mean
Number of samples 1289 2552 2472
Mean Y-Z 8.3 8.1 8.3 8.2
% of samples with Y-Z ≤ 8.5 70.6 76.3 69.2 72.0
% of samples with Y-Z 8.6-10.9 28.9 22.5 28.5 26.6
% of samples with Y-Z ≥ 11 0.5 1.2 2.3 1.3
Table 5.1 Percentage of IN samples collected in 2008, 2009 and 2010 with
yellowness Y-Z ≤ 8.5, Y-Z 8.6-10.9 and Y-=Z ≥ 11.
Clean wool colour of Merino sheep is moderately (0.29) [9] to highly (0.45) [8]
heritable and most Merino studs select for colour by subjectively assessing
their rams. Although measurements of fibre diameter are commonplace, few
wool growers use commercial services to objectively measure wool colour due
to the high cost of the test. Routine testing could identify sheep that produce
the brightest and whitest wool, and would be a more effective method for
selecting sheep on the basis of colour. Of equal importance is identifying wool
that is more resistant to photoyellowing.
The standard method for measuring the colour of scoured wool is IWTO-56-03
(Method for the measurement of raw wool colour) and there are many tests and
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84
standards to measure the lightfastness of textiles. Whilst a method to assess the
degree of photobleaching of coarse, loose wool fibre has been developed [60],
there is no test method available to measure the photoyellowing of fleece
wools.
Measuring the change in colour after exposure to light is relatively simple for
yarns and fabrics. These structures can be placed directly against the
observation port of a reflectance spectrophotometer to measure colour and the
fibres do not move relative to one another during the process of irradiation and
measurement. On the other hand, loose fibre must be contained in a
compression cell. The IWTO-56-03 method is unsuitable for measuring the
change in yellowness because the compression cell cannot be removed to
irradiate the sample.
This chapter describes the development of a new method to measure the colour
of wool fibre that uses commercially available, UV transparent cuvettes as
compression cells and complies with the key criteria of the IWTO-56-03
method for sample thickness and density. The method requires only 0.5 g of
fibre and uses a conventional reflectance spectrophotometer used for
measurements on textiles, whereas the IWTO method requires 5 g of fibre and
a special spectrophotometer for colour measurement. The new method
described here is suitable for measuring photostability and can be modified to
accommodate wet samples in order to investigate photo-initiated metal-
catalysed oxidation of proteins via Fenton type reactions, which requires an
aqueous medium.
5.2. MATERIALS AND METHODS
5.2.1. Preparation of fleece wool samples
In 2008, a total of 1289 wool samples were collected from the mid-sides of
one-year old Merino sheep (~100 g) from Sheep CRC Information Nucleus
flocks and were scoured and the colour measured by the Australian Wool
Testing Authority using the IWTO-56-03 method. Of these samples, 75
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85
samples having a broad range of initial yellowness were selected for this study.
Fleece wool from the same sheep was used as a control. The method used to
prepare and scour the wool is described in Table 2.1. The fibres were cut into
approximately 2 cm lengths, teased out and all visible vegetable matter was
removed.
For each sample, a mass of 0.512g 0.005 of fibre was evenly compressed to a
volume of 3.2 cm3 at a packing density of 160 kg/m
3 in a 4.5 ml
polymethylmethacrylate (PMMA) cuvette (Plastibrand, Wertheim, Germany).
Compression of the fibre was maintained by capping with a tight fitting 10
mm3 perspex cube (Figure 5.3a). The packing density was equivalent to the
requirement for the IWTO-56-03 test method. Cuvettes are available in
different materials: quartz, polystyrene (PS) and polymethylmethacrylate
(PMMA). Disposable PMMA cuvettes were selected for use as compression
cells because they are inexpensive, durable, transparent down to 300 nm
(Figure 5.1) and unaffected by exposure to UV. The thickness of the packed
wool complies with the IWTO colour test requirement that the sample
thickness should be a minimum of 8 mm to ensure that the wool layer
presented to the spectrophotometer for measurement is “infinitely thick”. The
wall thickness of the cuvettes is 1 mm which also complies with the test
method criteria that the cell material is preferably less than 3 mm.
CHAPTER 5
86
Figure 5.1 UV-visible absorbance spectra of poly methylmethacrylate (PMMA),
polystyrene (PS) and quartz cuvettes.
5.2.2. Colour measurement
The colour of dry wool samples was measured by placing the cuvettes over the
small area view (SAV) aperture (8 mm x 10 mm) in the inspection port of a
Gretag Macbeth Color-Eye 7000A spectrophotometer (Munich, Germany).
The IWTO test method recommends that the instrument port should be a
minimum of 10 mm smaller than the compression cell. However, the
difference can be less than 10 mm providing that the side walls of the cell are
not detected by the spectrophotometer during measurement. The cuvettes
comply with this criterion.
The spectrophotometer was configured with a D65 light source and a 10°
collection angle with the spectral component included (SCI). The CIE
tristimulus values X, Y and Z were measured by averaging two readings and
used to calculate the wool yellowness (Y-Z). The effect of PMMA on the
colour value can be corrected if required using IWTO standard ceramic tiles
directly against the observation port and behind PMMA. Regression analysis
indicates a linear relationship between the two measurements (R2 = 0.999). For
0
0.25
0.5
0.75
1
200 250 300 350 400 450 500 550 600 650 700 750 800
Ab
so
rba
nc
e
Wavelength (nm)
PMMA
PS
Quartz
CHAPTER 5
87
photostability measurements correction for PMMA was not required because
here we are only interested in the colour change for each sample obtained using
the same cuvette and spectrophotometer geometry.
5.2.3. Photostability of dry fibre
Wool strongly absorbs and photoyellows at wavelengths <380 nm due to the
production of yellow chromophores, and is photobleached by blue light (400-
460 nm) due to the destruction of these chromophores [59] (Figure 5.2).
Natural sunlight, including simulated sunlight from artificial sources,
comprises a broad range of wavelengths from UVB (285 nm) to the far infra-
red (2500 nm) and is therefore capable of inducing photoyellowing and
photobleaching in wool concurrently. The rate of yellowing of whiter wool,
which yellows from the outset, is therefore greater than yellow wool which
photobleaches before it yellows. Tests using simulated sunlight require
irradiation times of up to 10 days to induce a measurable colour change.
Photobleaching cannot occur when a UV light source is used and any colour
change is due to photoyellowing.
Figure 5.2 The action spectra of wool yellowing after exposure to an artificial light
source. Redrawn from [59].
0
10
20
30
40
50
60
320 340 360 380 400 420 440 460 480 500 500
Ye
llo
wn
es
s In
de
x
Wavelength (nm)
Initial wool yellowness
CHAPTER 5
88
A UVB light source is an appropriate for an accelerated test for photostability
of wool because these wavelengths (280-320 nm) photoyellow wool most
rapidly, and an appreciable degree of yellowing occurs after four hours
exposure. High energy UVB is absorbed by the outer surface of the cuticle
cells and induces yellowing and oxidation to an estimated depth of ~1-2 m
whereas lower energy UVA wavelengths penetrate wool fibre to a greater
extent and damage the cortex [2]. While the rate of yellowing in UVA is less
than for UVB, similar levels of photo-oxidation and yellowing are induced if
the period of UVA exposure time is increased to approximately 18 hours.
A lamp and sample holder assembly was manufactured by CSIRO Materials
Science and Engineering (Figure 5.3b) and was fitted with an extraction fan
and ventilation holes to maintain the operating temperature below 35°C,
thereby minimising the possibility of thermal yellowing of wool samples.
Cuvettes were placed in direct contact with a Philips TL20W/12RS (UVB)
light source (Figure 3.2) and irradiated for four hours.
Figure 5.3 (a) cuvette and cap (b) lamp and sample holder assembly.
b a
b
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89
5.2.4. Photostability of wet fibre
The UV dose required to induce a measurable colour change is less for wet
wool than for dry wool [2]. However the advantage of a shorter irradiation time
is offset by the additional processes of wetting and drying samples. Irradiating
wool while wet is essential to investigate if trace metals influence
photoyellowing via Fenton-type reactions because free radical and electron
transfer requires an aqueous medium in which to occur.
Wool fibre can be wet out to an even pickup prior to packing by saturating the
wool with a surfactant solution and removing the excess fluid using either a
centrifuge or a pad mangle. These methods were unsatisfactory because the wet
wool could not be teased out and packed evenly. Wetting wool after packing
was a more convenient method for handling a large number of samples and
injecting the solution into the cuvettes saturated the wool evenly.
Samples were wet out in cuvettes by injecting approximately 3 ml of non-ionic
surfactant solution in ultrapure water, using either 0.1% Leophen M (neutral
phosphoric acid ester with nonionic emulsifiers, BASF, Germany), 0.1%
Hydropol TN450 (nonylphenol ethoxylate, Huntsman Corporation, Australia)
or Teric G12A6 (alcohol ethoxylate, Huntsman Corporation, Australia). Unless
specified otherwise, wool was wet out with 0.1% Leophen M. The excess fluid
was removed by centrifuging so that the liquor to wool ratio was approximately
2:1.
The moisture content of wool under ambient and standard conditions has little
influence on the colour of wool [243], however optical effects make wet wool
appear to change colour. Therefore wool must be equilibrated at ambient
humidity prior to colour measurement. The percentage of moisture in wool
(regain) at a given temperature and relative humidity depends on whether the
wool was equilibrated after drying or after wetting [197] and it is
recommended that when measuring and comparing colour, all samples are
equilibrated from the same side [243].
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90
It is preferable and more efficient to dry the wool in cuvettes quickly because
equilibrating densely packed wet wool is prolonged under ambient conditions
and there is a risk of incubating micro-organisms which could discolour the
wool. After irradiation, the caps were removed and holes were pierced in the
bottom of the cuvettes. The onset of dryness was visualised by the colour
change from pink to blue of a similarly treated wool sample that was soaked
with a solution of cobalt chloride. The wool samples were thoroughly dried in
an oven at 80°C overnight and cooled to ambient temperature before replacing
the caps. Ideally, the drying conditions should be optimised to prevent thermal
yellowing while ensuring that the samples are adequately dried. Providing that
the samples are dried and equilibrated under the same conditions, colour values
can be compared. The degree of thermal yellowing of packed wet and dry wool
after drying overnight at 80°C increased the average of four Y-Z measurements
by approximately one unit and values can be adjusted accordingly if required
(Table 5.2).
initial dried at 80°C overnight
X Y Z Y-Z X Y Z Y-Z
Dry 62.95 66.64 62.60 4.0 61.94 65.63 60.45 5.2
Wet 62.55 66.23 62.38 3.9 61.91 65.81 60.78 5.0
Table 5.2 The average Y-Z of four wet and four dry wool samples before and after
drying at 80°C overnight.
Initially wool fibre was packed into PMMA cuvettes and irradiated with UVB.
The same mass of wet fibre irradiated with UVB for four hours and oven dried
did not yellow to the same extent as dry wool (Table 5.3). The effect was also
exhibited when wet wool fibre that was irradiated with UVB in quartz cuvettes,
indicating that the phenomenon was independent of cuvette material. In
contrast, wet wool that was exposed to a Philips TUV (UVA) light source
(Figure 3.1) for 24 hours (Table 5.3) yellowed more than dry wool.
The aromatic residues in the surfactant, Hydropol TN450 strongly absorb UVB
whereas Leophen M has a small UV absorption. Comparison of these
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91
surfactants with Teric G12A6, which does not absorb UVB, shows that the rate
of yellowing is unaffected by the absorbance properties of surfactant (Table
5.3) and that the surfactant is unlikely to cause the anomaly of the rate of
yellowing of wet wool fibre in UVB.
For comparison to fibre, pieces of wet or dry fabric were placed against the
inside transparent surface of a cuvette and packed with either wet or dry wool
fibre to make a total dry weight of 0.51 g. One set of samples was exposed to
UVB for four hours and the other set was irradiated with UVA for 24 hours.
The wet samples were oven dried and the yellowness was measured with the
fabric remaining in the cuvettes. The rate of yellowing of wet wool fabric was
greater after exposure to UVB compared to UVA and greater than dry wool.
Wet wool has been shown previously to yellow to a greater extent than dry
wool after exposure to UV radiation [2].
Surfactant Initial Y-Z of
dry wool
Y-Z after 4hr
UVB and
drying
Y-Z after
24hr UVA
and drying
Dry fabric# ― 6.4 7.9 9
Wet fabric# Leophen M 5.6 15.2 11.4
Dry fibre* ― 3.8 9.2 5.8
Wet fibre* Leophen M 3.6 9.9 ―
Hydropol TN450 3.6 8.5 11.6
Teric G12A6 3.5 9.7 ―
# average of two measurements
*average of four measurements
Table 5.3 The initial yellowness (Y-Z) of dry wool and the yellowness of dry wool
fabric and wool fibre that was irradiated in cuvettes, either wet or dry, with either
UVB for four hours or UVA for 24 hours.
5.2.5. Hydrogen peroxide assay
Hydrogen peroxide is produced when tryptophan is exposed to UV radiation
[244] and therefore this can also occur in photo-irradiated wool. Anomalous
differences in yellowness could be due to an increase in hydrogen peroxide
production and its bleaching effect. To examine this, liquor was extracted from
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92
wet wool in cuvettes that were either unirradiated or irradiated with UV. This
liquor was assayed to determine the hydrogen peroxide concentration.
Wool (0.5 g) was wet out at 100% pickup with a solution of non-ionic
surfactant (0.1% Leophen M), packed into cuvetttes and 2.0 ml of the same
solution was injected into the cuvettes. Two sides of the cuvette were exposed
to UVA for 24 hours each side or to UVB for 8.5 hours each side. After
exposure, the volume of fluid extracted was recorded and diluted to 3 ml. The
hydrogen peroxide content of the extract was assayed using the method
described previously [245]. T-test indicates that it is unlikely that there is a
difference in hydrogen peroxide production in unirradiated wool compared to
wool that was irradiated with either UVA for 48 hours (P=0.77) or UVB for 17
hours (P=0.52) (Table 5.4). However the test results are variable and it remains
possible that hydrogen peroxide production is greater in wet irradiated wool.
Hydrogen peroxide concentration of extract (ppm)
unirradiated UVB UVA
1 0.031 0.061 0.050
2 0.036 0.065 0.057
3 0.094 0.074 0.069
Table 5.4 Hydrogen peroxide concentration of fluid extracted from wet unirradiated
wool and wool irradiated with either UVB for 17 hrs or UVA for 48 hrs.
The reason for the lower yellowing of wet wool fibre compared to dry wool
after irradiation with UVB remains unclear. In the absence of a plausible
explanation the photostability of wet wool test specimens was measured using
a UVA light source for a longer period of time.
5.2.6. Statistical analysis
Data were analysed using the Microsoft Excel statistical package.
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93
5.3. RESULTS AND DISCUSSION
5.3.1. Comparison to the standard IWTO-56-03 method
The new method uses a tenth of the mass of wool required for the IWTO-56-03
test method. Smaller test specimens are an advantage when limited amounts of
sample are available and it is easier to remove all vegetable contaminants
which could otherwise affect colour values. Possible variations in colour values
associated with sub-sampling are reduced when the sample is thoroughly
blended (Table 5.5).
The relationship between the colour measured using the standard IWTO-56-03
method, and the new method using cuvettes as compression cells, was
determined by comparing the yellowness (Y-Z) of 75 samples from the IN
Flock. Differences of absolute colour values for the same sample are due to the
different configuration and calibration of the spectrophotometers. However, the
relationship between the Y-Z obtained using the two methods is linear
(R2=0.86) (Figure 5.4) and direct comparisons can be made.
Sample Number X Y Z Y-Z
1 62.95 66.76 63.92 2.8
2 63.12 66.94 64.06 2.9
3 62.18 65.96 63.21 2.8
4 61.11 64.81 62.12 2.7
5 63.06 66.89 64.15 2.7
6 62.92 66.73 63.74 3
7 63.14 66.95 64.06 2.9
8 62.82 66.64 63.83 2.8
Mean 62.66 66.46 63.64 2.83
Standard Deviation 0.70 0.74 0.68 0.10
Sample Variance 0.49 0.55 0.46 0.01
Range 2.03 2.14 2.03 0.30
95.0% confidence ±0.58 ±0.62 ±0.57 ±0.09
Table 5.5 Repeatability of yellowness (Y-Z) measurements using the new method.
CHAPTER 5
94
Figure 5.4 Comparison of the yellowness (Y-Z) of wool samples measured using the
new method with the yellowness measured using the standard IWTO-56-03 method.
The initial yellowness (Y-Z) of these samples is strongly correlated to the
change in yellowness (Y-Z) after irradiation (Figure 5.5) confirming the
results of a previous study that found that yellow wool yellows less rapidly
than whiter wool [59]. This effect is due to the saturation of the coloration
making the relative increase in yellowness less for wools that are already
yellowed, and is not due to the destruction of chromophores.
Figure 5.5 The relationship between the change in yellowness (Y-Z) with the initial
yellowness (Y-Z) for selected IN flock samples irradiated with UVB for 4 hours.
R² = 0.856
2
3
4
5
6
7
8
9
10
11
12
5 6 7 8 9 10 11 12 13 14 15 16
Y-Z
(N
ew
me
tho
d)
Y-Z (IWTO-56-03)
R² = 0.644
2
3
4
5
6
7
2 3 4 5 6 7 8 9 10
(Y
-Z)
aft
er
UV
B 4
ho
urs
Initial yellowness (Y-Z)
CHAPTER 5
95
5.3.2. Access to oxygen
Packed cuvettes require capping to guarantee that the wool is compressed to
the same density and to prevent wet samples from drying during irradiation.
Also, oxygen is required for photoyellowing to occur in both wet and dry wool
[3], and since the rate of photoyellowing is faster in wet wool, the oxygen
demand is likely to be greater [2]. To confirm that wool that is packed in a
cuvette has access to an adequate supply of oxygen, wet and dry test specimens
of a control wool were irradiated with UVB for four hours in cuvettes that were
either sealed or unsealed. Samples that were irradiated wet were not dried prior
to colour measurement. Paired T-test for both wet (P=0.44) and dry (P= 0.37)
indicate that there was no difference between the yellowness of wool irradiated
in sealed and unsealed cuvettes and that wool may have access to sufficient
oxygen for photoyellowing to occur. Comparison of photoyellowing in cells
with and without a flow of oxygen could confirm this. As a precaution,
cuvettes were stoppered with holed caps.
Sample Number Dry wool Wet wool
sealed unsealed sealed unsealed
1 9.18 9.31 2.90 3.01
2 9.30 9.77 2.38 3.79
3 9.69 9.42 3.04 3.46
4 9.12 9.50 3.75 3.22
Mean 9.32 9.50 3.02 3.37
Standard Deviation 0.26 0.20 0.57 0.33
Table 5.6 Yellowness (Y-Z) of wool in PMMA cuvettes before and after irradiation
for four hours in UVB.
5.3.3. Repeatability of the photostability test for wet wool
Six sub-samples from the same wool were used to determine the repeatability
of the test method to measure the photostability of wet wool after exposure to
UVA. Values of ± 0.44 for Y-Z are within the 95% confidence level (Table
5.7) and are acceptable.
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96
sample X Y Z Y-Z
1 54.48 57.79 45.23 12.56
2 52.78 55.95 43.74 12.20
3 55.60 58.90 45.56 13.34
4 55.24 58.58 45.54 13.04
5 56.14 59.60 47.09 12.51
6 54.74 58.03 45.57 12.46
Mean 54.83 58.14 45.46 12.69
Standard Deviation 1.17 1.25 1.07 0.42
Range 3.35 3.66 3.35 1.14
95.0% confidence ± 1.22 ± 1.32 ± 1.11 ± 0.44
Table 5.7 Repeatability of yellowness (Y-Z) measurements after irradiation with
UVA for 18 hours and drying at 80°C overnight.
5.4. SUMMARY
The use of PMMA cuvettes as compression cells provides a convenient and
repeatable method for measuring the colour of loose wool fibre that is similar
to the IWTO-56-03 test method. The cuvettes are also suitable for containing
wool fibre when measuring photoyellowing because the fibres are completely
restrained during irradiation and colour measurement.
A UVB light source is the most suitable for an accelerated method for
assessing the photostability of dry wool. For wet wool, it is recommended that
a UVA light source is used because the rate of yellowing of wet wool fibre
after exposure to UVB was less than dry wool fibre. This anomalous result was
independent of the wetting agent and the cuvette material, and the mechanism
has yet to be explained.
The new method for measuring the change in colour of dry wool after exposure
to UVB has been used to establish that the photostability of scoured wool is
moderately heritable (0.18 ± 0.10) and is positively correlated to whiteness and
softness [70]. The test could be used for routine colour measurement and to
identify sheep which produce the whitest and most photostable wools.
CHAPTER 6
97
CHAPTER 6. TRACE METAL CONTENT AND THE PHOTOSTABILITY OF FLEECE WOOL
6.1. INTRODUCTION
The colour and photostability of clean wool are heritable [70]. The estimate for
the heritability of photostability may be a second order effect and dependent on
the initial wool colour, since the change in yellowness of whiter wools is
greater than yellower wools [59]. However some other inherited property may
determine the propensity or resistance of fleeces to photoyellowing.
Differences in the photostability of wool may be due to heritable traits such as
the nature and distribution of UV-absorbing chromophores in the fibre, or the
presence of species that either protect or sensitise wool to photoyellowing. This
theory is feasible because the colour and thermal stability of wool subjected to
heat treatments is also heritable [246].
Copper and iron increase the photosensitivity [5] and photoyellowing of metal-
doped wool after exposure to UV wavelengths at levels as low as 22 mg/kg of
copper and 42 mg/kg of iron (Table 3.4). Since approximately 4% of the 700
wool samples collected from 408 individual sheep in the IN flock had either an
iron content ≥ 42 mg/kg or a copper content ≥ 22 mg/kg, it is possible that the
intrinsic levels of these metals in some fleeces contribute towards
photoyellowing, particularly in wet wool where photo-initiated Fenton-type
reactions can occur.
The aim of this study was to determine whether trace metals in wool,
particularly copper and iron, can account for differences in the extent of
yellowing of different wools after exposure to UV wavelengths. Since the
initial colour has a strong influence on the extent of photoyellowing, whiter
wools were examined using the test method described in Chapter 5.
CHAPTER 6
98
6.2. MATERIALS AND METHODS
6.2.1. Selection of samples
Mid-side wool samples were collected from Merino sheep in the Sheep CRC
IN flock and scoured. Although the tips of the fibre in these samples have
already been exposed to sunlight, the majority of the fibre has had minimal
exposure, particularly those parts that were close to the skin. The fibres in these
samples were cut during colour measurement at AWTA and were too short to
prepare fabrics or yarns for measuring photostability. Therefore the method
described in Chapter 5 was the most efficient option for preparing the samples
for assessing the colour change after exposure.
6.2.1.1. Evaluating the photostability of dry wool
The photostability of dry wool test specimens was measured at CSIRO
Materials Science and Engineering using the method described in Section
5.2.3. Those samples which were analysed for trace metal content (n=515)
were selected for this study.
The photostability measurements are stored on the IN Flock database by the
CRC for Sheep Industry Innovation.
6.2.1.2. Evaluating the photostability of wet wool
The whitest wool samples (Y-Z ≤ 7, AWTA, n = 85) that were analysed for
trace metal content were selected for this study. The samples had copper
contents ranging from 4.5–33 mg/kg and iron contents from 12–63 mg/kg.
Wool with the highest and lowest levels of copper and iron were included in
the sample set for comparison (36 samples). The photostability of wet sub-
samples was measured using the method described (Section 5.2.4) and the
results of duplicate test specimens were averaged. The trace metal content and
photostability measurements are tabulated in Appendix 1.
CHAPTER 6
99
6.3. RESULTS AND DISCUSSION
6.3.1. The effect of iron and copper content on photoyellowing
6.3.1.1. Photoyellowing of dry wool
Copper and iron content did not influence the extent of photoyellowing of dry
wool exposed to UVB for four hours (Table 6.1). Fenton-type reactions are
unlikely to occur in dry wool because metals require an aqueous medium to act
as catalysts.
dry wool irradiated with UVB 4 hrs
Y-Z ≤ 7 (AWTA) all samples
copper iron copper iron
R Square 0.002 0.07 0.0003 0.02
Standard Error 0.53 0.51 0.66 0.66
Observations 78 515
Table 6.1 Regression analysis results of the change in yellowness ( Y-Z) with iron
and copper content after irradiation of dry wool with UVB for four hours.
6.3.1.2. Photoyellowing of wet wool
It was anticipated that higher levels of copper and iron would increase the rate
of photoyellowing in wet irradiated wool, however, unlike metal-doped wool
(Table 3.4) the presence of these metals had no effect on white (Y-Z ≤ 7)
scoured fleece wools. The iron content did not influence either yellowness (R2
= 0.07) (Figure 6.1) or brightness (R2 = 0.07). Copper content did not influence
Y (R2 = 0.007) and the relationship with Y-Z was low (R
2 = 0.12) although
wool with higher levels of copper tended to yellow less. In addition, the copper
and iron content was not related to the rate of photoyellowing when wools
selected for extremes of metal content were included in the dataset (Figure
6.2). There are several possible reasons for the different effects of iron and
copper in scoured fleece wool and metal-doped wool.
CHAPTER 6
100
Figure 6.1 Iron content (mg/kg) of whiter wools and wool selected for extremes of
iron content versus the change of yellowness after exposure of wet wool to UVA for
18 hours.
Figure 6.2 Copper content (mg/kg) of whiter wools and wool selected for extremes
of copper content versus the change of yellowness after exposure of wet wool to UVA
for 18 hours.
5
6
7
8
9
10
0 10 20 30 40 50 60 70
(
Y-Z
) a
fte
r U
VA
18
hrs
iron content (mg/kg)
white wool
selected for metal content
R² = 0.12
R² = 0.16
5
6
7
8
9
10
0 5 10 15 20 25 30 35
(
Y-Z
) a
fte
r U
VA
18
hrs
copper content (mg/kg)
white wool
selected for metal content
white wool
selected for metal content
CHAPTER 6
101
Although the minimum level of copper and iron required to affect the
photostability of wool has not been established, it is possible that the levels of
copper and iron in the majority of the scoured wool samples were simply too
low to have any measurable effect. Only one sample of the whiter wools had a
copper content (33 mg/kg) similar to the lowest levels of copper-doped fabric
(Table 3.1) while the majority had copper contents ranging between 9 and 12
mg/kg. Six white wool samples had levels of iron greater than or equal to the
lowest level of doped wool while the majority had iron contents between 14
and 30 mg/kg. When including wools selected for metal content with the
whiter wools, 10 samples had copper levels between 20 and 77 mg/kg and 13
had iron contents between 40 and 65 mg/kg which are levels that are likely to
increase the rate of photoyellowing.
Another possibility for the greater effect of copper and iron in photo-irradiated
doped wool compared to scoured wool fibre is that metal ions in these are most
likely complexed to the fibre in quite different forms. The metal ions adsorbed
by wool under mildly acidic conditions become protein-bound hydroxides and
oxides [199]. Some of the adsorbed metals from doping solutions, and
exogenous metals in fleece wools, are likely to be bound to any available
carboxyl side chains [131, 132] or cystine residues [4] on non-specific proteins.
Metals incorporated into wool fibre during growth are likely to be bound to
specific proteins, particularly copper which is a constituent of an enzyme that
is associated with the keratinisation process.
In addition, approximately 30% of the copper and iron in doped wool is
unbound and can be easily removed by washing in non-ionic detergent (Table
2.3). On the other hand, loosely bound metals in clean fleece wools would have
been removed by the scouring process and the remaining metals probably exist
as metal-protein complexes.
The variation in the protein and lipid composition of the cuticle and cortex
[247] means that the nature of the metal-protein complexes will depend on the
location of the metals within the fibre. Although wool fibres may have similar
metal content, the density of metal ions is less when they are distributed evenly
CHAPTER 6
102
in the fibre compared to metals that are concentrated in the cuticle.
Although solvated metal ions are smaller than dye molecules and can penetrate
further into the fibre, the mechanism of diffusion is likely to be similar. Dyes
initially diffuse through the cell membrane complex between the overlapping
cuticle cells (Figure 6.3(a)), then migrate into the cuticle cells and the non-
keratinised inter-cellular cement [52, 248]. Successful dyeing is only achieved
after prolonged treatment at the elevated temperatures when the dye completely
penetrates the wool fibre. Diffusion into the fibre is incomplete with short
treatment times and at low temperatures, and the dye is concentrated in the
outer regions of the fibre (Figure 6.3(b)).
Whilst wool has a high capacity to adsorb metals from salt solutions [127, 249]
it is unlikely that metals are distributed throughout the entire fibre when wool
is doped with dilute, acidic (~pH 4) [131] solutions at room temperature [197],
and for relatively short treatment times. Metal binding sites are protonated in
acidified wool, and in dilute solutions, the migration of copper and iron into the
fibre may be restricted by the strong metal-binding capacity of thiols which are
in greater abundance in the cuticle. Metals adsorbed from doping solutions
have been found to concentrate in the cuticle [6].
Figure 6.3 (a) Pathway of dye diffusion into wool fibre [248] (b) Cross-section of a
wool fibre demonstrating the diffusion pathway of a fluorescent dye (CSIRO,
Materials Science and Engineering)
In contrast to doped wools, most of the copper is distributed relatively evenly
along the length of the fibre and throughout the cortex and the cuticle of native
cuticle cell
exocuticle
cortical cell
inter-cellular cement
endocuticle
(a) (b)
CHAPTER 6
103
wool fibre [108] and in human hair [129, 250] (Figure 6.4). The distribution of
other elements, including iron, is more affected by adsorption of metal ions
from exogenous contaminants than copper. The level of iron is higher in the
distal region of wool fibre (Table 4.10) and human hair (Figure 6.4) and is
greater in the cortex of wool [108], and the cuticle in human hair [124].
Figure 6.4 Distribution of iron and copper in human hair determined by Particle
Induced X-ray Emission (PIXE) [250].
Proteins in the cuticle have high levels of sulphur due to the cystine residues
[251] which are cross-linked and provide strength to the wool fibre. The
disulfide linkages cleave upon exposure to UV to yield cysteic acid [27] and
free thiols as cysteine [252] which can act as electron donors in metal catalysed
oxidation systems [83]. The influence of copper and iron on photoyellowing is
therefore likely to be greater when metals are concentrated in the cuticle. This
also could account for the different effect of copper and iron on the
photostability of metal-doped wool and fleece wools.
6.3.2. The effect of initial yellowness on photoyellowing
The relationship between the initial yellowness and the change in yellowness
of dry wool is largely dependent on initial colour (Figure 5.5) [59]. The
negative correlation was similar to wet wool exposed to UVA for 18 hours
although the regression coefficient was lower (R2 = 0.20) (Figure 6.5). The
CHAPTER 6
104
poorer correlation between the initial colour and the change in colour for wet
wool maybe because different mechanisms occur in wet and dry photo-
irradiated wool or that the range of values were different. The variance in the
photostability of both wet and dry wool may be due to genetic and phenotypic
differences in fleece wools.
Figure 6.5 The relationship between the initial yellowness and the change in
yellowness after exposure of wet wool to UVA for 18 hours.
6.3.3. Calcium, magnesium, manganese and zinc content and
photostability
The calcium, magnesium, manganese, sulphur and zinc contents of white wool
were not strongly correlated to the initial yellowness (Figure 6.6). Although the
change in colour after exposure of wet wool to UVA was not related to the
content of most metals, zinc content was negatively correlated to Y-Z (R2 =
0.25). The result is in contrast to wool doped with zinc which had similar
photostability as untreated wool (Table 3.3).
R² = 0.20
3
4
5
6
7
8
9
10
11
12
3 4 5 6 7 8 9 10
(Y
-Z)
aft
er
UV
A 1
8 h
ou
rs
Initial yellowness (Y-Z)
white wool high copper
high iron low copper
low iron white wool
all wool
R² = 0.20
CHAPTER 6
105
Figure 6.6 Plot of calcium, magnesium, manganese, sulphur and zinc content versus
the initial yellowness (Y-Z) and the change in yellowness after exposure of wet wool
to UVA for 18 hours.
The presence of zinc salts has been shown to reduce structural damage caused
by exposure to UV radiation of fibroblasts [253], skin cells in vitro and the
epidermis in vivo [254]. Zinc is not a redox active metal and cannot itself act as
an antioxidant. One theory for the protective effect of zinc in cells suggests that
zinc binds to and induces the production of metallothionein (MT) [255], a
family of cysteine-rich, low molecular weight proteins that captures superoxide
anions and hydroxyl radicals [256]. Another suggestion is that divalent zinc
displaces iron and copper from strategic sites on the protein, thereby preventing
metal-catalysed production of hydroxyl radicals. The latter mechanism is a
plausible explanation for protective effect of zinc on inert keratinised proteins.
However, the Pearson correlations of the zinc content with copper (0.06) and
iron (0.28) content were not negatively correlated in wool samples analysed in
this study. Alternatively, zinc could interfere with photo-induced electron
transfer or zinc oxide particles in fleece wool could act as a filter for UV.
In view of the evidence that zinc is associated with the immunity of sheep
[204], and that sheep which are resistant to fleece rot have wool with higher
levels of zinc [13], the Sheep CRC is conducting a trial in which sheep will be
injected with a multi-element supplement. The treatment includes zinc and its
effect on wool colour and photostability will be monitored.
Calcium Magnesium Manganese Sulfur Zinc
Y-Z
initial Y-Z
CHAPTER 6
106
6.4. SUMMARY
Unlike metal-doped fabric, increased levels of copper and iron in scoured wool
loose wool did not increase the rate of yellowing after exposure of wet wool to
UV radiation. Instead, wool with higher levels of copper tended to yellow less,
although the relationship between copper content and (Y-Z) was poor. It is
postulated that the different effects in scoured fleece wool and metal-doped
wool is due to the different regions and proteins in the wool fibre to which the
metal ions are bound.
The initial colour has a strong influence on the change in yellowness of dry
wool after exposure to UV wavelengths. The poorer correlation in wet
irradiated wool suggests that different reactions may occur in an aqueous
medium. Also, the variation in photostability for a given initial yellowness is
possibly a result of genetic and phenotypic differences in fleece wools.
The change in yellowness was negatively correlated to zinc content. This result
provides additional evidence that zinc may provide some protection from the
effects of UV radiation. Further study is warranted to confirm this finding and
determine the mechanism and efficacy of zinc in reducing the photoyellowing
of wool.
CHAPTER 7
107
CHAPTER 7. MEASUREMENT OF LIGHT PENETRATION THROUGH A SIMULATED MERINO FLEECE
7.1. INTRODUCTION
The aim of this study was to quantify the degree of light penetration from fibre
tip to base through fleece wool grown on the back of a Merino sheep and to
relate it to the trace metal content and the extent of damage along the fibre.
Measuring light intensity at the base of a fleece on a live sheep is difficult and
would require the implantation of a detector subcutaneously. It would be
possible to measure light at the base of the fibre by using the skin of a
slaughtered sheep. However, measurement using a simulated fleece is simple
and convenient and the influence of fleece density on light penetration can also
be assessed. It is possible to simulate a closed fleece which forms when the
fibres cluster into staples or an open fleece which represents the parts of the
fleece that separate as the sheep moves. Simulated fleeces were assembled
using wool staples from a Merino ram and the light intensity along the length
of the fibre was measured using a fibre optic probe.
The extent of photodegradation of the wool from a Merino ram was determined
by measuring the yellowness along the length of the scoured wool fibre and by
staining with methylene blue (CI Basic Blue 9). The damaged and oxidised
fibre tips have a greater affinity for the dye and are stained to a greater extent
than the root and mid-section [54]. Exposure of wool to sunlight during
measurements of light penetration was negligible and any photo-damage was
solely from exposure to sunlight while the wool was growing on the sheep. The
depth of tip damage was compared to the degree of light penetration through
the simulated fleece and the trace metal content along the length of the fibre.
CHAPTER 7
108
7.2. MATERIALS AND METHODS
7.2.1. Preparation of the simulated fleece
The density of wool required to prepare a simulated Merino fleece was
estimated from a typical wool sampling procedure. Mid-side sampling involves
taking approximately 100 g of greasy wool with an average staple length of 80
mm, from an area of approximately 250 250 mm from the mid-side of a
sheep.
A simulated Merino fleece was prepared using 18 g of approximately 100 mm
long wool staples collected from the fleece of a Merino ram. The fibre diameter
of the wool was 19.1m (SD 3.4m CV 17.9%). The staples were vertically
aligned and evenly distributed in a 95 95 95 mm plastic box lined with
densely woven black fabric that eliminated the transmission of light through
the bottom. A hole was pierced in the centre of the bottom of the box that
allowed a fibre optic probe to be positioned at any depth in the fleece. The
probe was encased in 10 mm diameter plastic tubing marked in 5 mm
increments. The tubing ensured that the probe travelled vertically through the
fleece. A second less dense fleece was prepared using 9 g of the same wool.
7.2.2. Measuring the depth of light penetration
The simulated fleece was exposed to either late spring or midsummer full
midday sunlight in Geelong, Victoria (Latitude: 38.18 °S Longitude: 144.34
°E) and the spectral intensity between 290–750 nm was measured
incrementally along the fibre using an Avantes AvaSpec 3648-USB2
spectrometer fitted with a FC-UV200 200 µm diameter fibreoptic cable probe
(Avantes BV, Erbeek, Netherlands). The probe was drawn down through the
fleece to ensure that wool did not become entangled in the probe assembly. The
wool was repositioned after the probe was initially drawn down for Trials 1 to
3, to simulate a closed fleece and prevent the formation of a tunnel through the
fleece which would allow more light to penetrate. The probe was held parallel
CHAPTER 7
109
to the fibres to estimate the penetration of light through the back wool of a
sheep.
The effect of reducing the fleece density on light penetration was measured in
Trial 3 after the mass of wool staples in the box was halved. The simulated
fleece was tilted to directly face the sun for Trial 4 to guarantee the maximum
possible exposure to light and the wool was left open as the probe was drawn
down to approximate those parts of the fleece on a live sheep that open as the
sheep moves. Two sets of measurements were taken for Trials 1–3 and these
were averaged. The percentage of total light was calculated using
measurements at 531 nm, which is the wavelength in sunlight having the
maximum intensity.
Trial No. Fleece size
(g/fleece) Exposure conditions Fleece type
1 18 midsummer sunlight closed fleece
2 18 late spring sunlight closed fleece
3 9 late spring sunlight closed fleece
4 18 midsummer sunlight open fleece
Table 7.1 Experimental conditions of trials to measure the penetration of light
through a simulated fleece.
7.2.3. Colour measurement and trace metal analysis
Greasy wool staples from a Merino fleece were cut into 10 mm segments along
the length of the fibre. Each bundle of segments were scoured separately using
conditions for maximum whiteness [160]. Scoured wool snippets were teased
out to remove all visible vegetable matter and the yellowness (Y-Z) was
measured using the new method for measuring the colour of small loose wool
samples described in Chapter 5 [257]. The tristimulus values X, Y and Z were
measured by averaging four measurements and used to calculate the wool
yellowness (Y-Z). The scoured segments were prepared and analysed for trace
metal content using the method described in Chapter 2.
CHAPTER 7
110
7.2.4. Staining with methylene blue to visualise damage
Scoured wool segments were immersed in a 0.1% solution of methylene blue,
thoroughly rinsed in water and air dried. The stained wool was compressed into
disposable cuvettes and the colour measured using the method described in
Chapter 5. Reflectance values were used to calculate the Kubelka Munk
coefficient (K/S) at 620 nm which quantifies the intensity of coloration from
staining and consequently the depth of tip damage.
7.3. RESULTS AND DISCUSSION
7.3.1. The extent of light penetration through a fleece
The percentage of the total direct sunlight that reached the base of a 100 mm
long simulated Merino fleece was 1% and approximately 6% at 60 mm from
the root end of the fibre (Trials 1–2) (Table 7.1).
Figure 7.1 The penetration of sunlight through a simulated fleece (% of total
sunlight).
0
20
40
60
80
100
0 10 20 30 40 50 60 70 80 90 100
% o
f to
tal
su
nlig
ht
at
53
0n
m
Distance from the tip of the fleece (mm)
Trial 1
Trial 2
Trial 3
Trial 4
CHAPTER 7
111
Halving the density of the fleece increased light penetration overall and at 60
mm from the base of the fibre, the lower fibre density allowed three times more
light to penetrate than the full Merino fleece. However the amount of light that
reached the root was 2% of the total, indicating that the base of the fibre is still
reasonably well protected in a fleece of lower density. The configuration used
in Trial 4 was intended to approximate the parts of the fleece on a live sheep
that open as the sheep moves. The extent of light penetration was greater under
these conditions than in a closed fleece and the intensity of light at 20 mm from
the root was approximately 20% that of the tip. The fleece provided a physical
barrier for light and there was little difference between the penetration of the
wavelength of maximum intensity (531 nm) and UV (290 nm) in sunlight.
7.3.2. The extent of damage along the fibre
For comparison to the light penetration data, the damage to the tips of a natural
Merino fleece was assessed by measuring the yellowness and the degree of
methylene blue staining along the length of the fibre (Figure 7.2).
Figure 7.2 Yellowness (Y-Z) of scoured wool and the intensity of the colour (K/S) of
Methylene blue stained Merino wool demonstrating tip damage.
0
0.5
1
1.5
2
2.5
3
3.5
4
3.0
4.0
5.0
6.0
7.0
8.0
9.0
0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 >100
K/S
Ye
llo
wn
es
s (
Y-Z
)
Distance from the tip of the fleece (mm)
Y-Z of scoured wool
K/S at 620nm of scoured wool stained with methylene blue
90-100
CHAPTER 7
112
The staples were randomly selected from a Merino fleece and therefore their
location on the sheep was unknown. Both yellowness and the intensity of the
blue colour from staining demonstrated that photo-damage extended to
approximately 40 mm from the tip of the staple and it has been shown by
measuring the light penetration through a fleece of similar density to a Merino
sheep that this represents the portion of the fibre that receives the maximum
dose of sunlight. The increase in yellowness at the base of the fibre is likely to
be from suint pigments or alkaline damage by suint.
This preliminary study demonstrates that the extent of light penetration and
damage to a simulated Merino fleece approximately 100 mm long can extend a
third of the length of the fibre and that the remainder of the fleece is protected
from exposure. Fleece density also influences light penetration. The depth of
damage is greater for more open fleeces which allow sunlight to penetrate
further [258] and this observation has been confirmed in this study. The root
and mid-section of a more compacted fleece are less damaged from sunlight
than the tips, whereas damage can extend further in a fleece that is more open
[27].
When staples form and the fleece is sufficiently dense, the portion of the fibre
which is close to the skin is well protected from photo-damage. Fleece density
is an important wool trait since it is genetically correlated to high fleece
weight, high clean scoured yield and fine fibre diameter [259] and it varies
with sheep breed [260]. Merinos have the fullest fleeces of all sheep breeds
with a density of 88 follicles per mm2
for a medium wool sheep (19.6–22.9 m
mean fibre diameter) [261] compared to Corriedale and Finn sheep with
approximately 20 follicles per mm2 [262, 263]. As well as reducing the
damaging effect of sunlight, a dense fleece affords protection from water
penetration [264] and decreases the extent of dust penetration [265]. Fleece
density is negatively correlated to wool yellowing in sheep exposed to high
humidity [217] although it can limit the rate of evaporation of suint in warmer
environments [266].
CHAPTER 7
113
Fibre length is also important and this study demonstrates that the amount of
light reaching the base of the fibres Merino fleece longer than 50 mm is likely
to be low. However, longer wools are more likely to retain moisture and have a
greater tendency to yellow than shorter wools which dry more quickly [267].
7.3.3. Trace metal content along wool fibre
With the exception of copper and zinc contents, which are marginally higher in
the tips compared to the remainder of the fibre, significantly higher levels of
most elements are found in section of fibre up to 40 mm from the tips (Figure
7.3). The extreme tips (0–10 mm) can have an order of magnitude more
calcium, magnesium, manganese than the base of the fibre and more
importantly much higher levels of iron (Table 7.2). It is likely that the iron
adsorbed from environmental contaminants and from aqueous doping solutions
is possibly bound to similar proteins and regions in wool. Therefore,
exogenous iron in the tips of the wool fibre could increase hydroxyl radical
production causing wool to yellow and become tender after exposure to
sunlight.
Figure 7.3 Trace metal content along the length of Merino wool fibres expressed as a
percentage of total trace metal content.
0
10
20
30
40
50
0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 >100
% o
f to
tal
co
nte
nt
Distance from the tip of the fleece (mm)
Zinc Copper
Iron Magnesium
Manganese Calcium
90-100
CHAPTER 7
114
Distance from
tip (mm) Ca Cu Fe Mg Mn Zn
extreme tips 0–10 1510 6.5 63 471 20 180
root 90–100 114 3.7 8 16 0.6 125
Table 7.2 Trace metal content (mg/kg) of the extreme tips (0– 10 mm) and the base
of wool fibre from a Merino fleece.
7.4. SUMMARY
The importance of reducing the damaging effect of sunlight is reflected by
efforts to develop methods to protect the growing fleece. Without protection,
the tips of the fleece will always be damaged by direct sunlight. Coating sheep
is an effective method to reduce the wool’s exposure to sunlight and dust.
Protecting fleeces with coats can increase the rating of style by one unit [72]
and make the wool whiter, cleaner and have less tip damage. Sheep which are
coated and shedded (Sharlea) yield wool that does not exhibit the same extent
of tip damage as pasture-raised sheep and the colour of the wool is relatively
consistent along the length of the fibre. Fleeces are adequately protected and
tip weathering is reduced if pasture-raised sheep are coated for a period of 6
months after spring shearing [268], after which time the fleece is sufficiently
long and dense to provide protection. However selection for fleece density and
ensuring that the fleece is as long as possible during summer are likely to
reduce the extent of damage along the fibre for grazing sheep.
This initial investigation into the relationship between light penetration through
a fleece and the extent of wool damage using a single simulated fleece is
limited. It demonstrates that the tips of the fleece, which receive the maximum
dose of solar radiation, also have high levels of trace metals that can increase
the damaging effect of UV wavelengths. However further studies are required
using a wider range of fleece types to understand the variability between sheep
and the effect of fibre diameter, staple length, wool wax, which assists in the
formation of staples, and other wool properties.
CHAPTER 8
115
CHAPTER 8. CONCLUSION
This study was initiated to expand the knowledge of the factors that affect the
colour of wool and was part of the Sheep CRC’s program to evaluate and
develop methods to produce the whitest wool possible. By improving its
colour, wool can compete with cotton and synthetics, thereby increasing the
demand for Australian wool.
The primary aim of the present research was to examine the role of trace metals
on the rate of yellowing during exposure to sunlight. The thesis was based on
studies of biological systems, in which protein-bound metals catalyse the
production of hydroxyl radicals, which then oxidise proteins. The enquiry was
also based on previous studies that identified that iron and copper can increase
the photoyellowing of doped wool, and that the presence of an iron chelator
reduces hydroxyl radical production in photo-irradiated wool.
The initial focus of this study was to develop a protocol to prepare large
numbers of wool samples for quantitative determination of bound metals. It is
essential that samples are rigorously cleaned in an identical fashion prior to
analysis to ensure the consistency of the results. Analysts use numerous
washing techniques for preparing samples, however these methods are not
standardised. The method described in this study was developed specifically
for processing large numbers of samples simultaneously and was validated
using standard hair samples (IAAA), a certified multi-element spectroscopy
standard, blanks and multiple analyses of a scoured wool fleece used
throughout as a control. The method was employed to prepare and analyse over
700 scoured wool fibre samples in this study, and 100 wool samples for a study
to examine the effect of sunlight and chelating agents on wool fleece wools.
The protocol could also be used for routine testing of large numbers of samples
of human hair or other animal fibres.
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116
The effect of trace metals on the colour and photostability of wool was initially
studied using fabric doped with copper, iron, manganese and zinc, which are
the most abundant transition metals found in wool. With the exception of zinc,
these metals have redox activity and can act as catalysts for the production of
hydroxyl radicals via Fenton-type reactions. Hydroxyl radical production in
photo-irradiated metal-doped wool was quantified using terephthalic acid as a
fluorescent probe.
Iron is the predominant catalyst in the metal-catalysed oxidation of proteins in
biological systems and hydroxyl radical production increased with iron content
in photo-irradiated wet wool. The presence of zinc had no effect on hydroxyl
radical production because it is redox inactive. Although it is a major catalyst
in biological systems, copper did not affect hydroxyl radical production.
Manganese reduced the production of hydroxyl radicals, possibly by
dismutating the superoxide anion or reacting with hydrogen peroxide to
produce molecular oxygen. Alternatively, manganese can inhibit the reduction
of Fe3+
to Fe2+
thus preventing iron from acting as a catalyst.
Some irradiated wools, particularly those doped with iron, were discoloured
and the Y-Z values did not reflect the differences in colour. Therefore, the
degree of photoyellowing of irradiated wool was determined using the Y-Z
values after washing with oxalic acid to remove unbound metal species, and
the brightness (Y) values.
In contrast to hydroxyl radical production, the rate of photoyellowing was
greater in copper-doped wool and increased with copper content. It is therefore
probable that hydroxyl radicals react with amino acids close to the copper-
protein binding site in preference to the fluorescent probe. The copper-binding
sites are possibly in close proximity to high concentrations of tryptophan or
tyrosine residues, which are known to form yellow products in irradiated wool.
This observation is worthy of further research.
Although more hydroxyl radicals were detected in iron-doped wools, these
fabrics photoyellowed less than copper-doped wools, suggesting that iron is
bound to different sites on the protein and that photoyellowing is more random.
CHAPTER 8
117
In addition, the anti-oxidant effect of manganese did not protect wool from
photoyellowing and manganese-doped wool yellowed to the same extent as
iron-doped wool.
The different effects of copper and iron were also demonstrated by the
differences in burst strengths of doped wool that was weathered by exposure to
an MBTF lamp while wet. The strength of iron-doped wool was less than
untreated wool, indicating an accelerated cleavage of structural proteins. In
comparison, the strength of copper-doped wet-irradiated wool was greater than
untreated wool which is consistent with protein cross-linking. This is also an
interesting finding worthy of further study.
The lack of correlation between hydroxyl radical production and the rate of
yellowing indicates that each metal is associated with different oxidation
mechanisms that occur in photo-irradiated wet wool. Although hydroxyl
radicals produced by metal-catalysed oxidation may not be the primary cause
of photoyellowing in wool, it cannot be guaranteed that the terephthalic acid
probe accurately quantified hydroxyl radical production in metal-doped wools.
The correlation between hydroxyl radical production with the burst strength of
irradiated iron-doped wool suggests that hydroxyl radicals are involved in bond
cleavage.
Wool samples were treated with buffered solutions at varying pH and the wet,
rinsed fabrics were exposed to UV to quantify the production of hydroxyl
radicals and the change in yellowness. Acidified wool yellowed to a greater
extent after exposure to UVB although the wool remained brighter. The
yellowness after irradiation decreased with pH whereas hydroxyl radical
production increased. Acidification did not remove metals, although other
species that could enhance hydroxyl radical production or species that quench
hydroxyl radicals may have been produced. This study highlights the
importance of the neutralisation process after dyeing under acidic conditions to
reduce photoyellowing, however this is offset by the increase in hydroxyl
radical production which could reduce the tensile strength of the fibre.
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118
To evaluate methods to reduce the catalytic effect of metals, a range of anti-
oxidants and chelating agents were added to the terephthalic acid solution prior
to the irradiation of metal-doped wool and their effect on hydroxyl radical
production was determined. Desferal, an iron chelator, effectively reduced
production of hydroxyl radicals and DTPA reduced the production to a lesser
extent. On the contrary, ascorbic acid, citric acid and EDTA increased the
production of hydroxyl radicals. Selection of reagents to reduce the effect of
trace metals in wool systems must therefore be used with caution because
chelates which do not occupy all the co-ordination sites on metals can have a
pro-oxidant effect.
Lipids readily oxidise to form peroxy radicals which damage proteins and the
reaction is increased in the presence of metal ions and a reducing agent. As a
preliminary investigation of their effect on photoyellowing and hydroxyl
radical production, the internal lipids were removed from wool prior to photo-
irradiation. Removal of lipids increased the concentration of hydroxyl radicals
suggesting the extracted species offers some protection as an anti-oxidant or
UV absorber, however the treatment did not affect the rate of yellowing. The
internal lipids could be the primary anti-oxidant species or the non-ionic
detergents that are used during the scouring process, and which are associated
with the lipids, could have some effect. Further investigation is warranted to
examine if the addition of certain lipid species to untreated wool will decrease
hydroxyl radical production because external lipids have been shown
previously not to have any photo-protective qualities. This present study was
not expanded to compare metal-doped wool with metal-doped lipid-depleted
wool because extraction of lipids did not affect the colour of photo-irradiated
wool, although comparison of these treatments would extend the knowledge of
the role of metals in wool.
Carbonyl species are by-products of protein oxidation by hydroxyl radical
attack and are used as a measure of the extent of oxidation. Carbonyls in
soluble proteins can be quantified colorimetrically by derivitisation with 2,4-
dinitrophenylhydrazine. Insoluble proteins such as wool require hydrolysis to
solubilise the protein structure, and this process can, itself, generate carbonyl
CHAPTER 8
119
species. Nevertheless this technique was used to assay carbonyl production in
metal-doped, photo-irradiated wool. Although the carbonyl content of wool
increased after exposure to UVB, the level of carbonyls was unaffected by the
presence of metals. This result may be due to the introduction of carbonyls into
the system from the hydrolysis process and the decomposition of amino acids,
or possibly other oxidative mechanisms may have a stronger influence on the
level of carbonyls in photo-irradiated wool.
After establishing the influence of copper and iron in doped wool, the next step
was to evaluate their effect in fleece wools. If photoyellowing catalysed by
these metals is the predominant cause of yellowing in fleece wools then one
would expect a positive correlation between copper and iron content with
yellowness. This was not observed in the 700 samples from sheep in the IN
flocks that were analysed in this study.
It is possible that the levels of copper and iron in fleece wools were too low to
have any effect because less than 4% of these wools had metal contents similar
to the doped wool in this study. In addition, the penetration of light through a
full fleece is limited and it is unlikely that the bulk of the fleece wool had been
exposed to sufficient sunlight to cause extensive photoyellowing. The catalytic
effect of copper and iron via Fenton-type reactions can only occur in wet wool
and it is also possible that the moisture content of the fibres is too low for these
reactions to proceed during wool growth on sheep.
While the copper and iron contents of fleece wools were unrelated to
yellowness, they were negatively correlated to brightness. The relationship was
independent of the yellowness value. This result is important because the
overall aim of this research is to produce bright white wool and removal of
metals would enhance the brightness of wool. The Glacial ™ scouring process
employs EDTA to remove residual metals from the fibre [269]. This process
was developed by the Wool Research Organisation of New Zealand (WRONZ)
to produce cleaner, whiter and brighter wool than conventionally scoured wool
and was achieved by removing the wool grease and dirt more effectively.
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120
The most significant observation in the examination of trace metals on fleece
wools was that the calcium, magnesium and manganese contents of yellow
wool were strongly correlated to the Y-Z value. Previous studies have shown
that suint contents can stain wool and that yellow discoloration is caused by
more alkaline suint. Although this is possible, it is also feasible that
Pseudomonas bacteria, which are ubiquitous on fleeces, could contribute to the
discoloration of wool and influence its calcium and magnesium content.
The yellowest wools, which had the highest levels of calcium and magnesium,
were sampled from one-year old sheep. Younger sheep have less mature
immune systems and produce less wax than older sheep making them more
vulnerable to infection by Pseudomonas bacteria. Pseudomonades require
calcium and magnesium to form stable biofilms that increase both the adhesion
of bacteria to substrates and the cohesion between cells. The high calcium and
magnesium content of heavily yellowed wool may be from residues of biofilm.
Alternatively, wool from yearling sheep, which has higher levels of calcium
and magnesium from milk feeding, may encourage the growth of
Pseudomonas. This observation requires further investigation because 1.5% of
over 6000 wool samples from IN flock had unsatisfactory Y-Z values ≥ 11, and
identifying the cause of the extreme yellowness will enable measures to be
taken to prevent it.
A method was developed to measure the photostability of small scoured wool
samples in order to further examine the effect of copper and iron in fleece
wools. This step was required because the major portion of wool taken from
the mid-side of sheep would not have been exposed to the maximum dose of
sunlight and sufficient moisture to allow Fenton-type reactions to occur.
The method was used to examine the photostability of the whitest wool
samples because the rate of photoyellowing is dependent on initial colour.
However, selection based on colour limited the range of metal contents and the
number of samples with higher levels of copper and iron. Additional samples
with extremes of copper and iron content were therefore included for
comparison. The level of copper and iron did not influence the rate of
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121
yellowing of wet wool after exposure to UVA radiation, most likely because
the intrinsic levels of these trace metals in wool were too low. Zinc content was
negatively correlated to the change in yellowness and this observation requires
further investigation.
Using the developed method and wool samples from IN flocks in 2008, the
Sheep CRC established that the photostability of wool is moderately heritable.
This work will be expanded to samples collected in 2009 and the results will
identify which animals produce the whitest and most photostable wool.
Photoyellowing may occur at a faster rate depending on where the metals are
located within the fibre, and this distribution may be highly significant. The
metals in doped wool may be mainly restricted to the cuticle, whereas those in
natural wool may be more evenly distributed throughout the fibre. It would be
interesting to confirm this theory using Synchrotron X-ray fluorescence
(SXRF), a technique that has been used successfully to image trace metals in
human hair and plant tissue.
This study has shown that the major portion of a 100 mm simulated fleece is
well protected from sunlight and dust contamination. The study was limited,
however it demonstrated that the tips of wool fibre on sheep receive the
maximum dose of sunlight and have higher metal content than the remainder of
the fibre. While iron and copper was not observed to have an effect in whole
fibre, it is feasible to assume that metals in the fibre tips can accelerate
photoyellowing and photo-tendering, similar to the metals in doped wool,
particularly when the fleece is wet from rain. Coating a fleece until it is
sufficiently long has been previously shown to improve wool quality, and this
has been confirmed in the present study. However, further research is required
to evaluate the fleece properties that affect the degree of sunlight and dust
penetration and the variability of these properties between sheep.
There were limitations to sampling whole fibre samples from IN flocks. The
shearing protocol and timing were different for each IN site, which meant that
wool had been exposed to the environment for different periods of time. In
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122
addition, the sheep were grazed on different pastures and some were treated
with products that contaminated fleeces with zinc and possibly other metals.
Therefore, the contribution of each effect of climate and nutrition could not be
assessed, especially when analysing whole fibres.
The factors which affect wool’s colour are numerous and the trace metal
content of fleece wool is a result of a complex sequence of biochemical
reactions and events that affect the geochemistry of soil and water. Therefore,
maintaining experimental sheep in a controlled environment with more rigidly
defined nutrition and husbandry practices would help to identify the most
important influences on the colour and trace metal content of fleece wool.
The demonstrated effect of copper and iron on the colour and strength of
photo-irradiated doped wool shows that these metals can significantly affect
the quality of wool during processing and the garment after laundering. Some
tap waters contain high levels of metals which are adsorbed by wool. The
adsorbed metals can increase the rate of photoyellowing and photo-tendering
during laundering, particularly when the garment is exposed to sunlight while
damp.
If wool is to compete with cotton and synthetics in the next-to-skin knitwear
market for which bright fabrics are essential, it is important to produce the
whitest wool and maintain its colour during growth on the sheep and during
processing and laundering of the garment. Identifying the contribution of trace
metals to the colour and photostability of wool is a step towards understanding
the requirements for producing bright white and brightly coloured photostable
wool garments.
123
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147
APPENDIX 1 Photostability of wool after exposure to UVA for 18 hours
while wet and the trace metal content (mg/kg)
Sheep Genetics ID Ca Cu Fe Mg Mn Zn Initial Y-Z delta Y-Z
26IN082008080160 313 9 28 17 2.3 122 4.1 7.3
26IN082008080296 115 7 28 4 1.1 119 3.5 8.4
26IN082008081209 213 9 26 11 1.6 109 3.7 8.1
26IN082008080173 44 5 22 2 0.4 110 3.9 8.1
26IN082008080362 92 11 63 4 1.1 113 4.0 8.5
26IN082008081006 258 14 36 14 2.5 117 4.0 7.1
26IN082008080182 298 9 20 21 1.7 135 4.3 7.6
26IN082008080381 184 6 22 10 1.5 122 3.8 6.4
26IN082008080122 98 10 30 5 1 123 3.6 7.5
26IN052007070422 240 14 30 0.4 1.4 138 3.6 6.5
26IN052007070469 327 8.7 24 26 1.5 127 4.0 6.6
26IN052007070412 173 7.7 29 11.1 1.3 123 4.2 7.5
26IN012008080781 66 8.7 13 3.3 0.6 110 3.7 8.3
26IN052007070532 125 13 22 4.4 0.9 126 4.0 8.0
26IN012008080829 186 9 21 10 1.7 125 3.6 7.9
26IN052007070466 170 17 24 5 0.8 163 4.4 6.3
26IN012007070432 52 4.5 22 5.85 0.7 126 3.4 8.1
26IN052007070459 182 9 42 13 2 113 4.1 8.0
26IN052007070422 380 8.8 47 35 2.6 125 4.1 7.4
26IN012008080684 240 12 35 15 2.7 134 3.8 7.4
26IN012008080565 79 10 12 3.6 0.4 130 3.9 7.1
26IN052008080369 46 10 46 3.1 0.6 113 4.2 7.4
26IN052008080427 91 11 21 3 0.7 117 4.4 7.1
26IN052008080290 41 12 18 1.6 0.4 133 4.0 6.7
26IN052008080406 114 10 25 4.2 0.8 147 4.6 5.9
26IN062007077713 206 11 36 6.8 1 146 4.1 6.3
26IN012007070389 111 7.7 17 6.5 1.1 93.5 4.0 8.9
26IN052007070202 91 6.7 24 5.6 1 114 4.1 7.8
26IN052007070509 543 8.8 22 57 2.1 129 4.0 7.5
26IN082007070793 134 6.7 23 7.8 0.7 139 3.4 6.9
26IN012008080796 65 8 23 5 0.7 126 3.6 8.5
26IN012008080957 124 9 25 6 1.4 126 4.0 8.4
26IN042008081482 54 7.1 24 5.6 1.8 120 3.8 6.6
26IN072008080511 115 10 30 11 1.3 130 4.4 7.6
26IN042008081474 640 13 14 56 9 97 4.4 7.8
26IN012008080693 53 12 13 2.2 0.4 110 3.5 8.8
26IN012008080324 64 10 13 3 0.5 120 3.7 8.6
26IN012008080262 550 15 18 30 2.2 120 4.2 6.9
26IN052007070412 58 12 30 0.6 0.5 138 4.2 6.1
26IN052008080413 39 9 23 2.7 0.4 129 4.3 7.5
148
APPENDIX 1 Photostability of wool after exposure to UVA for 18 hours
while wet and the trace metal content (mg/kg)
Sheep Genetics ID Ca Cu Fe Mg Mn Zn Initial Y-Z delta Y-Z
26IN062007077429 56 8 22 0.9 0.4 168 3.9 6.4
26IN052008080423 108 14 28 3.2 0.8 135 4.4 6.3
26IN012007070440 202 8.3 14 15.5 1.2 111 3.5 8.8
26IN012007070436 375 6.2 21 37.5 2.5 124 3.7 8.5
26IN012007070653 101 5.9 13 6.5 0.9 142 3.6 8.1
26IN012007070333 204 6 46 19 1.8 100 3.9 8.2
26IN052007070391 217 9.9 56 17.5 3.1 144 4.2 6.7
26IN082007070230 189 9.2 28 9.7 0.8 134 3.6 8.0
26IN012008080170 44 12 17 3 0.4 112 4.0 9.3
26IN012008080686 188 8 25 11 1.7 98 4.0 8.5
26IN042008081474 75 8.6 20 4.7 2.1 120 3.7 8.6
26IN042008081540 109 10 16 5 4.2 106 4.1 8.2
26IN042008081452 132 9 17 6 3.2 101 4.2 7.4
26IN042007071402 71 12 14 4 3.2 134 3.7 6.6
26IN052007070241 79 13 20 4.2 0.6 122 4.2 7.5
26IN052007070133 54 12 22 2.1 0.4 121 3.7 8.2
26IN052008080282 41 10 18 2.3 0.4 132 4.3 7.1
26IN052008080414 221 12 20 8 1.1 148 4.1 6.5
26IN012007070717 45 6.4 19 2.65 0.5 119 3.7 8.6
26IN012007070095 288 5.4 15 25 1.8 107 3.7 9.2
26IN012007070347 187 7.3 16 14 1.6 129 3.6 8.9
26IN012007070354 63 7.3 39 6.8 0.9 119 4.0 8.2
26IN042007070601 77 8.5 18 5.55 2.5 106 3.9 8.7
26IN042007071188 54 7.8 15 4.45 2.1 125 3.6 8.3
26IN052007070262 156 7 21 10.3 1 108 3.4 8.3
26IN062007077296 294 7.8 13 15.4 0.3 126 3.8 8.7
26IN062007077713 190 8.5 15 9.5 0.3 111 3.8 8.0
26IN012008080654 97 7 13 5 0.7 127 3.5 9.6
982 000093564363 158 12 15 7 1.2 118 3.6 8.5
26IN072008080401 75 11 22 8 1.1 132 4.1 7.9
26IN042008081588 46 13 16 2 2 112 4.2 7.5
26IN042008081421 177 11 19 9 4.6 92 4.3 8.0
26IN042008081447 111 7 13 5 3.1 96 4.3 7.3
26IN052007070533 57 12 22 0.4 0.3 113 3.8 6.8
26IN052007070198 146 15 21 2.8 0.6 125 3.7 6.5
26IN052007070605 31 12 17 1.2 0.1 103 4.0 8.5
26IN052008080246 74 7 25 3 0.7 125 4.4 7.3
26IN062007077295 36 11 14 0.4 0.1 135 3.9 8.7
26IN042007071163 41 7.1 13 2.8 1.8 113 3.8 8.1
26IN042007070595 182 8.6 20 11.5 5.3 106 4.2 8.3
26IN052007070405 187 7.2 37 15 1.4 97.5 3.8 7.6
149
APPENDIX 1 Photostability of wool after exposure to UVA for 18 hours
while wet and the trace metal content (mg/kg)
Sheep Genetics ID Ca Cu Fe Mg Mn Zn Initial Y-Z delta Y-Z
26IN052007070326 109 8.6 33 8.1 1.4 131 4.2 7.1
26IN052007070378 408 9.1 22 39.5 1.7 130 3.7 8.1
26IN082007070939 70 11 23 3 0.6 98 4.5 7.8
26IN082007070792 123 33 27 6 1.2 108 4.5 7.0
26IN042008081513 340 77 17 23 4.5 120 5.0 7.3
26IN082008080255 629 46 9 53 2.2 108 5.4 6.3
26IN072008080159 150 30 24 8.3 1.5 110 5.6 5.2
26IN082008081230 769 28 15 73 3.6 110 6.1 6.5
26IN082008080061 116 28 19 3 0.6 100 5.0 6.3
26IN082007070134 386 28 31 22 2.5 100 5.0 7.8
26IN082007070410 192 27 12 5 0.6 77 5.0 7.5
26IN072008080440 210 27 16 11 2.1 120 5.1 7.5
26IN082007070410 311 5.9 7.6 21.8 0.6 99 4.1 8.2
26IN082007070090 63 4.9 10 5.3 0.2 124 4.4 7.9
26IN032007000853 55 4.9 12 4.9 1.7 98 4.2 9.7
26IN032007000877 302 4.7 9 17 8.9 97 5.5 8.1
26IN032007000829 73 4.6 13 5.2 3.3 82 6.3 8.0
26IN032007001110 39 4.5 11 2.7 1.4 104 4.1 8.0
26IN032007000917 53 4.2 8.8 3.3 1.4 90 5.2 8.1
26IN032007000646 63 4.2 13 6.3 2.3 96 4.0 8.8
26IN032007001253 371 4.1 11 30.5 9.6 113 7.2 7.3
26IN032007000974 43 3.8 8.8 2.2 0.8 84 5.0 8.7
26IN032008080075 73 11 65 14 3.8 100 4.8 8.5
26IN022008080628 130 13 61 10 4.1 145 4.4 8.2
26IN052007070438 215 8.4 59 32.3 1.9 133 4.2 8.4
26IN052007070332 111 6.8 53 12 1.4 180 4.7 7.3
26IN022008080628 54 7.3 52 11 2.4 130 4.9 7.5
26IN052007070330 279 7.2 51 26.1 1.9 130 4.2 7.7
26IN012008080781 47 7.2 20 3.8 0.4 120 4.3 8.2
26IN072008080492 270 45 36 20 3.4 145 4.5 6.8
26IN012008080324 57 8.2 17 5.5 0.8 130 4.3 7.3
26IN012008080565 36 7.5 17 3.3 0.5 130 4.4 7.8
26IN022008080294 845 8 10 74 7.9 125 9.5 4.8
26IN062007077792 51 5.6 8.9 3.8 0.1 98 3.9 7.5
26IN012007070473 31 7.5 8.6 1.3 0.2 100 4.5 8.2
26IN012007070623 430 8.9 8.4 26 1.2 91 7.1 6.6
26IN012007070547 59 6 8.4 2 0.3 93 5.5 8.5
26IN082007070123 366 6.2 8.1 28.4 0.7 124 4.2 6.7
26IN032007000888 536 5.3 8 46 13 122 6.6 7.7
26IN012008080164 700 10 7.7 43 1.2 99 5.7 7.7