1. A MASTER THESIS REPORT O
MECHA ICAL CHARACTERIZATIO OF HYBRID BIOCOMPOSITES
Master thesis report submitted to the Department of Chemistry at the Technical University of
Munich, the Faculty of Chemistry and Pharmacy of the Ludwigs-Maximilians-University of
Munich and the Department of Mathematics and atural Science of the University of
Augsburg in the partial fulfillment of Masters Degree in Advanced Materials Science
Under the guidance of:
Prof. Dr. Wolfgang W. Schmahl
Department of Earth and Environmental Sciences
Ludwig Maxmilians University of Munich,
Munich, Germany
Submitted by
Ganesh Kumar Tirumalasetty
Advanced Materials Science
Technical University of Munich, Ludwig Maxmilians University of Munich,
University of Augsburg
ovember 2007-July 2008

2. CO TE TS
Acknowledgement 1
Abstract 3
Chapter: 1 – Introduction 4
Chapter: 2 – Literature review 7
2.1 – Importance of nanocomposites 7
2.2 – Shell structures and their importance 9
2.3 – Influence of shell design 11
2.4 – Shell composition and microstructure 12
2.5 – Shell mechanics 14
Chapter: 3 – Experiments and Testing Procedures 17
3.1 – Materials 17
3.2 – Specimen preparation 20
3.3 – Mechanical characterization tests 22
3.4 – Cost effective device for measuring mechanical
properties of intricately shaped bio-composites
40
Chapter: 4 – Results and Discussions 46
4.1 – Results 46
4.2 – Discussions 72
Conclusions 77
Engineering Perspective 78
Future Scope 79
References 80

3. 1
Acknowledgement
I am extremely pleased to present before you my Master thesis report titled
Mechanical Characterization of Hybrid Biocomposites. I consider it a privilege to express
a few words of gratitude and respect to all those who guided and inspired me in completing
this thesis.
I am extremely thankful to Prof. Dr. Wolfgang W. Schmahl, Lehrstuhl für
Anorganische und Biogene Geomaterialien, Ludwig Maxmilians University of Munich for
being kind enough for permitting me to carry out this work and for providing me with
necessary facilities for my project. His invaluable guidance and enduring association
throughout the course of this internship has encouraged me a lot.
I would like to take this opportunity to extend my gratitude to Dr. Erika
Griesshaber, Ludwig Maxmilians University of Munich for the SEM work and for giving her
valuable suggestions during the entire course of project.
My heartfelt thanks are due to Mrs. Enders and Prof. Dr. P. Gille, Ludwig
Maxmilians University of Munich for their guidance and keen interest in sample preparation.
I would like to take this opportunity to extend my gratitude to Prof. Dr. Steinhauser
and Mrs. Christine Hausner Henzel of Hochschule Muenchen for their impact testing
equipments, Mr. Thomas Wehlus of University of Augsburg for the Nanoindentation testing,
Mr Pondicherry Shanmugham Kartik of University of Ulm for the Microindentation
testing, Dr. Stefan Eichhorn and Mr. Tobias Winkler of Technical University of Munich for
their three point bending tests and Fracture Toughness measurements and Dr. Enrico
Schwabe of Bavarian State Collection of Zoology (ZSM) for the mollusc samples and also for
his help in finding the names and literature of the molluscs from New Zealand .
My cordial thanks are due to Mr. Casjen Merkel, Ludwig Maxmilians University of
Munich and Mr. Srihari Subramanian, Technical university of Munich for their valuable
guidance and suggestions course of project.

4. 2
On my personal behalf, I would also like to thank Mr. Markus Sieber and his group
members from the mechanical workshop and Mr. Max Haeberle and Mr Deblef Koerner
from electronic workshop, Ludwig Maxmilians University of Munich for constructing the
tensile testing machine.
My project work would remain incomplete without acknowledging the moral support
of all the research group members of Prof. Dr. Wolfgang W. Schmahl, as the knowledge
delivered from them has been a stepping stone and a moral rider for this project.
Finally it is said that behind every successful man there is a women and in my case it
happens to be my mother. I would like to express my deepest thanks for the support provided
to me by my mother Mrs. Jayalakshmi Tirumalsetty and my father Mr. Lava Kumar
Tirumalsetty without which my work would not have moved so far.

5. 3
Abstract
Structural composite materials found in nature like the mollusc shells posses a unique
combination of mechanical properties to protect soft tissues from the mechanical aggressions
of outside environment. These shells are composed of calcium carbonate crystals interleaved
with layers of viscoelastic proteins, having dense, tailored structures that yield excellent
mechanical properties. However, mechanical testing of these samples in a natural condition
hasn’t been done before owing to the intricate shape of these materials. Studies have been
done till date, using micro and nanoindentation in dry conditions. Although there are few
studies on elasticity and strength, yet characterizing these materials in wet conditions hasn’t
been performed so far. The main basis for entire research is that the molluscs originally live in
water and the mechanical properties of them could be influenced by water.
Initially a cost effective tensile testing machine was designed and constructed
indigenously to serve the purpose of testing these intricately shaped biological composites.
The tensile testing equipment is relatively inexpensive, requires little skill to construct and is
easy to use and further yields sufficiently accurate data on mechanical properties and its use
for studies of shells of different sizes requires only minor modification. For the first time,
three-point bending tests were performed to determine the elastic and ultimate properties of
two different kinds of mollusc in wet and dry conditions. While dry specimens exhibit brittle
behaviour, hydrated specimens exhibited matrix-reinforcement-pullout ductility. The flexural
modulus, bending strength, fracture strain and nominal work-to-fracture properties were
higher for the hydrated samples rather than the corresponding dehydrated samples.
Also impact toughness and fracture toughness measurements were carried out on the
shell samples in wet and dry conditions and the samples show relatively higher toughness in
wet conditions than their dry counterparts. This implies that it is the structure itself that is
mainly responsible for these mechanical properties. The fracture behaviour and morphologies
of the fracture surfaces varied significantly with the influence of water on these shell
materials.
Key words: Molluscs, Wet, Dry, mollusc, three point bending, Impact, Fracture Toughness.

6. 4
Chapter-1
Introduction
Billions of years of evolution have produced extremely efficient natural materials,
which are increasingly becoming a source of inspiration for engineers. Biomimetics the
science of imitating nature is a growing multidisciplinary field which is now leading to the
fabrication of novel materials with remarkable mechanical properties. These high-
performance natural composites are made up of relatively weak components arranged in
intricate ways to achieve specific combinations of stiffness, strength and toughness. If one is
able to determine the features which control the performance of these materials one could
introduce these features into artificially bio-inspired synthetic materials, using innovative
techniques such as layer-by-layer assembly or ice-templated crystallization. The most
promising approaches, however, are self-assembly and biomineralization because they will
enable tight control of structures at the nanoscale (Zhiyong et al., 2003). In this 'bottom-up'
fabrication, also inspired from nature, molecular structures and crystals are assembled with a
little or no external intervention. The resulting materials will offer new combinations of low
weight, stiffness and toughness, with added functionalities such as self-healing.
Recent investigations have shown that mollusc shells are natural nanocomposite
materials, composed of layers of nanocrystalline inorganic aragonite (CaCO3) surrounded by
an organic biopolymer matrix in an arrangement commonly described as a ceramic plywood.
Their laminated structure achieves a thousand fold increases in toughness over its constituent
materials (Kamat et al., 2000). The current methods for synthesis are not very practical,
because the micro architectures and toughening mechanisms have not been explored and
understood completely (Kessler et al., 1996). This deficiency opens up this field of
nanocomposites for further research and development. Therefore these inexpensive light
weight structures have been inspiring material scientists to develop synthetic, biomimetic
nanocomposite assemblies that attempt to reproduce nature’s achievements (Sellinger et al.,
1998). Investigations in this field can also gain considerable commercial importance owing to
the substantial usage of nanocomposites in aerospace and automobile industries and their
constant search for composites of light weight, high strength and low cost still remains to be
explored.

7. 5
Although these materials exhibit extraordinary combinations of mechanical properties,
macroscopic mechanical measurements like tensile tests have been rarely performed on these
materials due to their intricate shapes. Better understanding of the know-how of bulk
properties of these biomaterials in relation to the microstructure can be had by measuring the
mechanical properties at micron and nanometer scale (Li and Nardi, 2004). Several studies
have been done till date, using microindentation and nanoindentation to estimate the hardness
of the sea shells in dry and embedded conditions ( Griesshaber et al., 2006, Perez-Huerta et
al., 2007, Merkel et al., 2007). Nevertheless, characterizing these materials in wet conditions
hasn’t been performed so far due to the difficulty in characterizing the indentations on the
sample surface. In addition, measurements taken in wet conditions can cause damage to the
sample surface. Consequently micro and nanoindentations were carried out on dry and
embedded samples.
Previous studies on bones and teeth signify that testing in dry and embedded condition
is not ideal and wet condition would be closer to the in vivo conditions experienced by these
materials (Blackburn et al., 1992, Weaver 1996). Biological samples show a significant
change in mechanical properties in a fluid environment compared to the usual ambient testing
conditions. Hardness and elastic stiffness values usually increase with decreasing water
content and the degree of change is considerable (Schöberl and Jaeger 2006, Dall’Ara et al.,
2007, Bell et al., 2008). Unlike the other biomaterials, mollusc shells with their hierarchical
structure also should show similar behavior in terms of their exposure to the humid
environments.
Further, testing of these samples in a natural condition hasn’t been done before owing
to the difficulty of sample preparation and area determination for standard mechanical testing.
Three-point bending tests were performed to determine the elastic and ultimate properties of
two different kinds of molluscs in different positions of the shell in wet and dry conditions.
Additionally classical toughness measurements like the Charpy Impact toughness and fracture
toughness have also been carried out in wet and dry condition to confirm the influence of
water on the mechanical properties of these shell materials.
In this study, three mollusc shells were tested with nanoindentation, microindentation
and three point bending techniques. The aim of this study is to investigate the differences in

8. 6
mechanical properties of the shell materials in wet and dry conditions, evaluated by
nanoindentation, three point bend tests, impact and fracture toughness testing.
Several complex and rather expensive electronic instruments have been designed by
scientists until now to measure the strength of general engineering materials like metals,
alloys, ceramics, polymers and composites. Generally these instruments are not readily
available to biologists studying the mechanical properties of these shells. Further installing
tensile testing equipment or testing samples statistically large number of samples is relatively
costly. Keeping the above points in view, a tensile testing machine is indigenously built which
is relatively inexpensive, requires little skill to construct and is easy to use and further yields
sufficiently accurate data on mechanical properties and its use for studies of shells of different
sizes requires only minor modification.

9. 7
Chapter-2
Literature Survey
2.1- Importance of nanocomposites
Nanocomposites of organic and inorganic materials are a fast growing area of research
focused mainly on the ability to obtain control of the nanoscale structures through innovative
synthetic approaches. The properties of nano-composite materials depend not only on the
properties of their individual parents but also on their morphology and interfacial
characteristics (Nanocomposites, internet, 2008).
Fig 2.1: anocomposites and its commercial applicability in the field of automobile,
space, ocean, medical, agricultural, automotive, building construction, railway, etc
Experimental work has generally shown that virtually all types and classes of
nanocomposite materials lead to new and improved properties when compared to their
macrocomposite counterparts. This rapidly developing field is generating many exciting new
materials with novel properties by not only combining properties from the parent constituents
into a single material but also to produce properties which are entirely different from those of
parent materials. Therefore, nanocomposites promise new applications in many fields such as
mechanically reinforced lightweight components.

10. 8
The general class of organic/inorganic nanocomposites may also be of relevance to
issues of bio-ceramics and biomineralization in which in-situ growth and polymerization of
biopolymer and inorganic matrix is occurring. Finally, lamellar nanocomposites represent an
extreme case of a composite in which interface interactions between the two phases are
maximized. Since the remarkable properties of conventional composites are mainly due to
interface interactions, the materials dealt here could provide good model systems in which
such interactions can be studied in detail using conventional bulk sample as opposed to
surface techniques. By judiciously engineering the polymer-host interactions, nanocomposites
may be produced with a broad range of properties. Inorganic layered materials exist in great
variety. They possess well defined, ordered intralamellar space potentially accessible by
foreign species. This ability enables them to act as matrices or hosts for polymers, yielding
interesting hybrid nano-composite materials.
Lamellar nano-composites can be divided into two distinct classes, intercalated and
exfoliated. In the former, the polymer chains alternate with the inorganic layers in a fixed
compositional ratio and have a well defined number of polymer layers in the intralamellar
space. In exfoliated nano-composites the number of polymer chains between the layers is
almost continuously variable and the layers stand greater than 100 Å apart. The intercalated
nano-composites are also more compound-like because of the fixed polymer/layer ratio, and
they are interesting for their electronic and charge transport properties. On the other hand,
exfoliated nano-composites are more interesting for their superior mechanical properties.
Presently, most of the work is focused on the lamellar class of intercalated
organic/inorganic nanocomposites and namely those systems that exhibit electronic properties
in at least one of the components. This subclass of nano-composites offers the possibility of
obtaining well ordered systems some of which may lead to unusual electrical and mechanical
properties. Selected members of this class may be amenable to direct structural
characterization by standard crystallographic methods. An important issue in this area is that
there is very less information available regarding the structural details, and therefore, any
system that is subjected to such analysis is of great interest. Nanocomposites also offer the
possibility to combine diverse properties which are impossible within a single material, e.g.
flexible mechanical properties and superconducting properties. Another exciting aspect is the
possibility of creating heterostructures composed of different kinds of inorganic layers, which
could lead to entirely new area of multifuntional materials

11. 9
2.2- Shell structures and their importance
Natural selection provides a tool by which nature can process, improve, and refine
biologically based organisms over millions of years. Scientists can learn from these
evolutionary refinements and develop technologies based on natural designs. At present, even
the simplest bio-mineralized structures cannot be synthesized in the laboratory without the use
of living organisms. Hence studies are being carried out with the intention to contribute to the
ongoing development of the next generation of synthetic materials (Sarikaya, 1994,
Srinivasan et al., 1991), based on biomimickry.
Many natural materials exhibit extraordinary combinations of mechanical properties
which are achieved through highly tailored and organized hierarchical microstructures. In
particular materials which function as natural body armor such as mollusc shells, possess a
structure with important features and properties at a variety of length scales, from the various
constituent blocks to the overall integrated and synergistic mechanical model of their complex
assemblies. Basic inorganic materials used in nature are, on their own, very weak. However,
when combined with proteins, self-organized into highly ordered structures, and refined over
long periods, these basic materials make very strong composites, sometimes increasing their
strength by orders of magnitude (Kamat et al., 2001).
Examples of such biological materials include bone, teeth, sponge spicules, diatoms,
and mollusc shells where their nanometric dimension play an important role in their superior
mechanical behavior (Vincent, 1991, Weiner, 1997). These complex composites contain both
inorganic and organic components in their macro-, micro-, and nanostructures (Baer et al.,
1992, Lowenstam, 1989). The complexity of these structures and their ability to self-assemble
has drawn considerable attention (Nicolis and Prigogine, 1997, Sarikaya 1994, Whitesides,
2002). An increase in strength due to structure can be seen in other laminates as they form
stronger materials from weak base materials, however, the relative strength gain found in
these biocomposites remains unparalleled in synthetic materials. By investigating various
shells with similar composition, but dissimilar structural organization, one can observe the
role of macro-, micro-, and nanostructures in the mechanical response of such biocomposites.
Such unique structures have been widely studied, stimulating the creativity of materials
scientist for developing new synthetic materials exhibiting high feat of properties.

12. 10
One of the most biogenic composite studied is the mollusc shell, especially the
nacreous structure. It is composed by platelets of aragonite (calcium carbonate) stacked in a
brick-and-mortar fashion. The crystals are glued by a fibrous 0.1–5% organic phase
(Srinivasan et al., 1991). Such a layered structure has been on the basis of the high bending
strength and toughness of nacre.
Fig 2.2: Schematic drawing of the cross-lamellar structure of Stombas gigas. Each layer
also consists of first-, second-, and third-order lamella (Lin et al., 2006).
It is assumed that these materials possess extraordinary mechanical properties because
of extensive delamination at large distances ahead of the crack tip, and by the adhesive
properties of the organic matrix. The nacreous organic matrix is complex in nature and is just
one of the seven different microarchitectural structures found in molluscs’ shells (Weiner,
1997). The most common is the crossed lamellar structure that may present four hierarchical
levels of organization (Lowenstam, 1989). Those various levels may be a way found by
nature to reduce the anisotropy that exists at the lowest ordering stages (Nicolis and
Prigogine, 1997). The tendency of reducing mechanical anisotropy has been found in other
mineralized tissues like the skeletons of sea urchins, lamellar bone or biogenic silica and has
been linked to the fact that the material must perform satisfactorily under many different
loading conditions (Nicolis and Prigogine, 1997).

13. 11
2.3- Influence of shell design (Ketchum, internet, 2008)
1. There are only a few basic structural systems for post and beam structures, but for
shell structures, there are thousands, each requiring a unique approach to design.
2. The supports for a shell are more important than the shell. Stiffest path concepts
are useful in understanding shell structures.
3. Shell structures are very complex and carry forces by many paths. Shell structures
are usually understood as a set of beams, arches and catenaries and can be
analyzed by that approach.
Fig 2.3 : a) Arch Bridge b) Fuselage of aircraft
4. Shell structures can carry relatively large point loads. For any shell structure, there
will be a simple method of analysis that can be used to check the more precise
analysis.
5. Shell structures get their strength by shape and not by high strength of materials. A
typical example would be that of an egg shell which is easier to compress and
break in the horizontal direction but in the vertical direction it takes relatively
higher compressive load to break it into fragments.
Fig 2.4: Egg shell in compression
6. Shell structures, because of their complexity and unfamiliarity require a large lead
time for developing the design. Shell structures are also light weight constructions
and find applications in structural engineering deigns like fuselages in aeroplanes,
ship and boat hulls and also the roofing’s of buildings
a b

14. 12
2.4- Shell composition and microstructure (Prezant, 1998)
The molluscan shells are comprised of calcium carbonate mineral which is either
deposited or embedded within an organic matrix. The organic matrix constitutes to less than
10 % of the shell and is mainly composed of glycoprotiens. Inorganics like aragonite and
carbonate constitute towards 90 % of the shell and form various microstuctural layers in the
bivalvia.
Fig 2.5: a) Fibrous prismatic b) acreous (malacsoc, internet, 2008)
The most primitive shell types had prismatic nacreous microstructure. In this shell
type, a prismatic layer composed of either calcitic or aragonitic prisms of variable thickness
underlies the periostracum. This, in turn is underlain by a nacreous or mother of pearl layer
that forms the inner shell surface. The prisms, often polygonal in cross section, are arranged
perpendicular to the shell surface and the flat nacreous tablets lie in the plane as the shells
surface. Various types of prismatic layers can be discerned including simple, fibrous,
composite and spherulitic prismatic layers. A specialized simple prismatic layer, the
myostracum, forms the distinct shell structure involved in the muscle attachment. As such
myostracum lies underneath most of the muscle attachment areas and is surficially revealed as
muscle scars of the shell interior surface.
The nacreous layer of aragonite forms the lustrous inner layer of many mollusk shells
having the composition similar to that of pearl. Nacre can be deposited either polygonal or
round tablets that merge with growth within and upon the organic matrix. In general, nacre
grows as
• thin flat sheets called sheet nacre
• tall pyramidal columns called columnar nacre
• Elongated stacked tablets called row stacked nacre.
a
b

15. 13
In many pteriomorphia, a foliated calcite layer is present composing of flat elongated
lathes or blades that represent roofing shingles. Most bivalves have shells with some crossed
lamellar microstructure. As such there could be variations on the theme of rectangular
lamellae rods or blades crossing each other. Small third order lamellae, essentially elongate
crystals compose the larger second order lamellae which in turn contain still larger first order
lamellae. The first order lamellae, often rectangular in form are deposited parallel to the shell
surface. Many pteriomorphs have first order lamellae’s arranged either concentric to or
parallel to the shell edge with alternating blocks arranged obliquely to each other. Complex
cross lamellar microstructures can have first order lamellae arranged in cones that lie
perpendicular to the shell surface. Most hetrodonts have complex cross lamellar layer often
found below a foliated calcitic layer.
Fig 2.6: a) Simple prismatic b) Crossed lamellae (malacsoc, internet, 2008)
Small spherules of aragonite may compose a distinct homogeneous layer found in the
protobrachia and some heterodonta. A homogeneous layer sensu stricto is composed of small
spherules whereas a granular homogeneous layer is composed of larger granules.
Although shell microstructure is conservative for the most part, significant alterations
in basic microstructures could take place with the shifts in the environment.
a
b

16. 14
2.5- Shell mechanics
The mathematically regular spirals of mollusc shells (Thompson, 1942, Cortie 1992,
Cortie 1993) and their relatively higher resistance to fracture (Wang, et al., 2001, Li, et al.,
2004) have been of interest since long time. The mechanical strength has resulted from the
evolutionary interplay between predation on the species and its need to ensure survival and
productiveness (Vermeij, 1993). The fracture toughness of Inorganic CaCO3 is 0.9 MPa√m,
which is similar to ordinary glass (Ashby and Jones, 1980). However, natural materials like
mollusc shells are vastly more durable and tougher than man made materials like glass.
Research has slowly revealed the reasons.
Firstly, shells are composite structures of aragonite, other calcareous materials such as
calcite, where 95 volume % of the composite is the inorganic material and up to 5% by
volume of protein ‘glue’ known as conchiolin. A large component of the toughening derives
from the unique aragonitic microstructure of the nacre layer which facilitates crack tip
blunting, deflection, closure and bifurcation—concepts well known from the field of fracture
mechanics. Moreover, it has been found that the nacreous structure can also undergo inelastic
deformation (Wang et al., 2001), which is helpful. Finally, it has recently been claimed that
aragonite platelets themselves are nanostructured in such a way as to permit some internal
plastic deformation (Li et al., 2004). When a crack travels perpendicular to the layers of
bricks, it deflects around the aragonite crystals, which then must pull out as the crack widens
(Okumura and de Gennes, 2001)
Nacre, the inner lustrous layer of many mollusc seashells is a material that has
intrigued scientists for many years. The structure of this material has evolved through millions
of years to a level of optimization not currently achieved in engineered composites. Materials
scientists have spent many years analyzing the structure of the aragonitic calcium carbonate
layers, their crystallography, defect structure and also growth mechanisms (Katti, Katti,
2006). Likewise, much work has also been done on analyzing the nature of the protein rich
20–30 nm layers that separate the aragonitic platelets (Katti, Katti, 2006). The organic matrix
has been known to contain aspartic acid rich macromolecules. These macromolecules have
been linked to control of crystal nucleation and growth, texture and morphology (Weiner and
Addadi, 1997, Mann et al., 1989). The role of the constituents of the organic phase: h-chitin,
silk-like proteins and acidic glycoproteins are to control the mineralization of aragonite.

17. 15
Recently, cryo-transmission electron microscopy has been used to evaluate the
structure of organic layers in bivalve Atrina embedded in vitrified ice. These experiments
have indicated that the organic layers are composed of highly ordered and aligned h-chitin
fibrils (Kalisman et al., 2001). Over several decades, many spectroscopy techniques have
been used to study the crystallographic, molecular and electronic structure of nacre. Fourier
transform infrared spectroscopy has been used to analyze the organic and inorganic layers of
nacre (Katti, Katti, 2006).
Considerable work has also been done on the experimental determination of
mechanical properties of nacre. Measurements based on careful experiments were first done
by Currey in 1977. Mean values of tensile strength, compressive strength, bending strength
and modulus of elasticity were obtained for several species. Currey’s paper from 1977 also
gave a stress– strain curve of nacre in tension. This curve indicated that nacre shows a linear
(elastic) region until a sharp yield point at about 0.2% strain followed by failure at about 0.6%
strain. Further Jackson et al. obtained the mechanical properties of nacre for species Pintada
umbricata . They reported a modulus of about 70 GPa and a tensile strength of about 170 GPa.
They also reported a work of fracture of 350–1240 J/m2
. Sarikaya et al. 1992, measured
fracture strength of red abalone (Haliotis rufescens) to be about 185 MPa and fracture
toughness of 8 MPa√m. Recently, we have reported three-point bend tests on samples of
nacre from red abalone shells. SEM micrographs were obtained across the cross-section of the
samples from the compression to tension faces. Distinctly different microstructures were
observed for the compression and tension faces. It is found from micrographs that the
interface where the transition appears to occur from compression to tension is about a third
from the top of the sample.
These biocomposites displays a high fracture toughness which is comparable to those of
some high-technology structural ceramics (Jackson et al., 1988, Currey, 1977, Sarikaya et al.,
1995). Several toughening mechanisms of the nacre have been examined in the literature and
exploited to produce strong materials like (Sarikaya et al., 1995, Cleg, 1999, Rao et al., 1999)
1) crack blunting/branching,
2) micro crack formation,
3) internal stresses

18. 16
4) mechanical properties of proteins on dissipating the fracture energy (Smith et al.,
1999)
Fig 2.7: A model of biocomposites. (a) A schematic diagram of staggered mineral
crystals embedded in protein matrix. (b) A simplified model showing the load-carrying
structure of the mineral–protein composites. Most of the load is carried by the mineral
platelets whereas the protein transfers load through the high shear zones between
mineral platelets (Gao et al., 2003).
The crossed lamellar microarchitecture of the shell provides for channelled cracking in
the outer layers and uncracked structural features that bridge crack surfaces, thereby
significantly increasing the work of fracture, and hence the toughness, of the material ( Kamat
et al., 2000). The coarsest structures are the inner, middle, and outer layers, which are oriented
in either weak or tough orientations with respect to the direction of potential catastrophic
crack propagation. Sequential cracking of the weak and strong layers of the shell occur during
crack propagation, and it is necessary to partition the energy dissipated during fracture into
these two processes. This is done here through quantitative micromechanics modelling
(Kamat et al., 2004). These nanocomposites also exhibit a generic mechanical structure in
which the nanometer size of mineral particles is selected to ensure optimum strength and
maximum tolerance of flaws (Gao et al., 2003).

19. 17
Chapter-3
Experimental and Testing Procedures
3.1- Materials
This study involves testing and analysis performed on a three shell specimens. The samples
are from three different genus of mollusc called Dosina anus, Gari strangeri and Elliptio.
Dosina anus and Gari strangeri was collected in Paihia, New Zealand. Elliptio is a fresh
water mollusc collected from the Ohio River, Illinois USA.
3.1.1- Dosina anus (Powell, 1979)
Fig 3.1: Dosina anus from ew Zealand.
Description:
These shells are relatively large, solid and moderately inflated and winged posteriorly as
shown in the fig 3.1. The ridges are flat-topped, fine and concentric. There are number of
these ridges which are approx 19-20 per cm and there is no lamellose at the dorsal margins.
Pallial sinus is broadly V shaped and its apex is slightly beyond the middle of the shell. They
are mostly white in colour with some having a light yellowish brown colour externally.
Habitat
These shells are found in Northern Islands and Cook Straight in shallow waters especially on
the coastal beaches.

20. 18
3.1.2- Gari strangeri (Powell, 1979)
Fig 3.2: Gari stangari from ew Zealand.
Description:
These shells are elongated in shape with a distict posterior flexure. They are slightly rostrate
with the lower edge being slightly pointed out. They have a sculpture of closely spaced sharp
and crisp concentric lamellae of about 30 per cm distance. There colour ranges from cream to
buff with a slight iridescence as shown in the fig 3.2. The interior of the shell has colour
ranging from cream to chrome yellow.
Habiatat
These shells are mainly found on the north and south islands of Australia and New Zealand.
They are commonly seen in shallow water off ocean beaches.

21. 19
3.1.3- Elliptio Crassidens (Mussel Manual, internet, 2008)
Elliptio is a fresh water mollusc locally abundant in some parts of the Ohio, Illinois and
Indiana. Because of its shape, it is commonly referred to as Elephants ear, Mule's ear or blue
ham.
Fig 3.3: Elliptio crassidens from Ohio River, Pulaski County, Illinois.
Description
The shell in general is heavy, solid, and triangular with dark brown to black periostracum.
The Anterior end of the shell is rounded and the posterior end is pointed with dorsal margin
having a slight curvature (fig 3.3). In general younger shells have a curved ventral margin
whereas in older shells it is mostly straight. Umbos are low and usually not elevated above the
hinge line. The Posterior ridge is prominent and is angled sharply and the surface of the shell
is mostly smooth. Periostracum is reddish brown with faint green rays in small shells,
becoming dark brown towards the left valve. The lateral teeth are short, rough, and straight
and the beak cavity is very shallow. The colour of nacre usually varies from purple to pink or
white.
Habitat
Elliptio shells are mainly found in large rivers in mud, sand, or fine gravel.
Present Status
These shells are relatively widespread in other parts of the world but are a rarity in the
Midwest. They are locally abundant in some parts of the Ohio and White rivers of Illinois and
Indiana. Although they are available now, there is a growing concern about the dangers of
extinction in Missouri, Ohio, Wisconsin and Illinois.

22. 20
3.2- Specimen Preparation
anoindentation sample preparation
Samples were prepared by cutting macroscopic fragments of 5 mm thick and 25 mm in length
with a diamond saw. Further the samples were polished with increasingly fine sand paper
number up to 4000 grit along the transverse direction. Thereafter the surface was polished by
means of a napped cloth impregnated with 1 µm diamond paste. A scratch was created on the
sample surface from one end of the sample to the other end. The intersection of edge of the
scratch and the outer edge of the sample is taken as the reference point and the indentations
are taken at a distance of 1500 µm in Y direction and 1000 µm in X direction. The distance
between the indents is fixed at 15 µm. Rewetting the samples was performed by placing the
samples for 60 hrs in tap water. To avoid the scatter in the hardness for dry and wet
conditions, the indentations in wet conditions were taken in the close proximity (30 µm) of
the indentations in dry condition.
Fig 3.4: Sample preparation for three point bending test a) Wire saw used for cutting
the samples b) sample cutting and numbering
Specimen Preparation for Three point bending test
The shell specimens were initially cut into small fragments using a High-speed water-cooled
diamond saw or a wire saw along the longitudinal1
direction. All the four specimen surfaces
except the top and the bottom faces were ground and polished to 2.5 mm breadth and 25 mm
length. The polished samples are numbered in ascending order from anterior end to posterior
end (i.e from left to right) so that one could remember the positions of the samples easily as
shown in the fig 3.4 b. The actual lengths of the samples are 25 mm but the guage length (i.e
length between the supports in three point bending test) is 15 mm.
1
Note: longitudinal direction here means that the cutting is done along the direction of layers and transverse direction here
refers to the cutting being done along the direction perpendicular to the direction of layers of the shell.
a
b
Anterior
end
Posterior
end

23. 21
Specimen Preparation for impact toughness tests
For classical impact tests one requires samples which are relatively flat and long in order to
place the samples in between the grips of the impact testing machine. Consequently, Elliptio
crassidens was used in impact toughness tests. The shell specimens were initially cut into
small fragments using a wire saw along the longitudinal direction. The samples are then
grinded and polished to the dimensions of 6*4*50 mm3
(i.e. 6 mm breadth 4 mm height and
50 mm length). All the samples are finely polished to 10 µm surface finish, so that the crack
does not initiate from the surface of the sample. In general for impact toughness
measurements a notch is introduced into the sample, but in the case of our testing since most
of the tests are for comparative purpose, the samples were tested without creating a notch.
Fig 3.5: Sample preparation for Fracture toughness tests
Specimen Preparation for Fracture toughness tests
In the case of fracture toughness tests one requires samples which are relatively flat and thick
in order to test the samples in between the three point bending supports of the tensile testing
machine. Thus, Elliptio crassidens was used in impact toughness tests. The shell specimens
were initially cut into small fragments using a wire saw along the longitudinal direction. The
samples are then grinded and polished to the dimensions of 2.5*5*35 mm3
(i.e. 2.5 mm
breadth 5 mm height and 35 mm length2
). All the samples are finely polished to 1 µm surface
finish by polishing it with a diamond paste, so that the crack does not initiate from the surface
of the sample. A notch is introduced into the sample by means of a High speed cutting saw.
At the tip of the notch, a fatigue crack was initiated by tapping a single edged razor blade into
the notch with a hammer while the specimen was constrained in a bench vise to avoid the
crack propagation. The schematic diagram of fracture toughness samples and their dimensions
are shown in fig 3.5
2
Note: Although the sample has the length of 35mm the guage length here was 20 mm.

24. 22
3.3- Mechanical Characterization tests
3.3.1- anoindentation
Nanoindentation is the method of measuring hardness and other micromechanical properties
of metals and materials and Nanoindenter is a tool which has an appreciable contribution in
enabling us in the understanding of material properties at nano length scales. The device is
used to measure mechanical properties like Elastic Modulus, hardness, toughness, fatigue, etc.
of materials, by means of indentation and scratch tests.
The basic setup of the Nanoindenter consists of an indenter tip usually made of diamond
formed into a sharp pyramidal shape like the three sided Berkovich indenter, an arrangement
of sensors and actuators to apply and measure the indenter load and displacement. In the
advanced testing equipment, the nanoindenter is integrated with high resolution microscopes
for qualitative analysis as well as electrical testing units for measuring micro electrical
properties of materials. There are different kinds of setups even based on the testing methods
viz. direct testing and differential testing with the latter being quicker in measuring apart from
the fact that it avoids the need of a lot of correction factors (Fischer-Cripps, 2004).
Working of a anoindenter
Basically, indentation is made by applying a small load (less than 100 mN, and using Hysitron
instruments, we can get to loads in the range of micro newtons (µN)) with a diamond indenter
of a known geometry and the change in displacement as a function of load as the indenter is
pressed into the specimen surface is recorded. A schematic of the load-displacement curve is
given below in Fig.3.6. Once a nanoindentation machine has collected load-displacement
data, a number of different analyses can be used to determine the mechanical properties of the
sample from the data.
Fig 3.6: Schematic of a typical load-displacement curve

25. 23
Nanoindentation tests were performed using NanoTest 600 testing system. This system
records the dynamic load and also displacement of the indenter with a Berkovich three sided
pyramid with a force resolution of 100 nN and displacement resolution of 0.06 nm. A
significant advantage of using the "depth sensing indentation" approach is that one can obtain
much more information than just hardness. However the results obtained depend on the
analysis model chosen and can be very sensitive to the details of the analysis.
The slope of the curve, dP / dh, upon unloading is indicative of the stiffness S of the contact.
This value generally includes a contribution from both the material being tested and the
response of the test device itself. The stiffness of the contact can be used to calculate the
reduced modulus of elasticity Er as
where A(hc) is the area of the indentation at the contact depth hc (the depth of the residual
indentation), and β is a geometrical constant on the order of unity. The reduced modulus Er is
related to the modulus of elasticity Es of the test specimen through the following relationship
from contact mechanics.
Here, the subscript i indicates a property of the indenter material and ν is Poisson's ratio. For a
diamond indentor tip, Ei is 1140 GPa and νi is 0.07. Poisson’s ratio varies between 0 and 0.5
for most materials (though it can be negative) and is typically around 0.3.
Nanoindentation hardness is calculated using the indented peak load (Pmax) and projected
area of contact (A) of the indentation. From the load–displacement curve, hardness can be
obtained at the peak load as
H =Pmax /A
The displacements and loads can be calculated in various ways, for instance, the former using
a differential capacitor gauge or an LVDT (Linear Variable Differential Transducer), and
some advanced equipment use Lasers to measure the displacement (similar to that in AFM),
S = dP / dh

26. 24
and loads on the other hand, can be controlled using electromagnetic or controlled using
electromagnetic or electrostatic attenuation or actuation using springs.
Modes of operation of the anoindenter
There are many modes of operation of the Nanoindenter based on the property of interest we
are looking for, for instance:
• Basic Hardness Mode
• Fatigue Test Mode
• Creep Mode
• CSM Mode (for Fracture toughness)
• Scratch Test Mode
and so on and so forth (Fischer-Cripps, 2004)…
Basic Setup of anoindenter
Given under in Fig 3.7 is a photograph of a Nanoindenter.
Fig 3.7: Photograph of a nanoindenter ( anoTest 600)
Models in anoindentation
Models give us an idea and an understanding of the theory and assumptions behind the
working and functioning of the system. Nanoindentation too is based on models, and given
under is a brief description of the most basic model.

27. 25
Oliver and Pharr Model (Oliver and Pharr, 1992)
Oliver and Pharr (O&P) made a critical improvement to the method proposed by Doerner and
Nix (D&N). Sneddon's (Sneddon, 1965) contact solution predicts that the unloading data for
an elastic contact for many simple indenter geometries (sphere, cone, flat punch and
paraboloids of revolution) follows a power law that can be written as follows
P = α hm
In this equation P is the indenter load, h is the elastic displacement of the indenter and α and
m are constants. Oliver and Pharr apply this formulation to determine the contact area at
maximum load as it is valid even if the contact area changes during unloading. To do this,
they derive the following relationship for the contact depth from Sneddon's solutions
hc = hend – θ*(Pmax/ S)
where q = 0.72, 0.75 and 1, for cone-, sphere- and flat-punch-geometry respectively.
The procedure for O&P analysis is to then fit a power law function to the unloading segment.
This yields the contact stiffness as slope of this function at maximum load. This slope in
addition to the appropriate value of q is used in order to determine the actual contact depth so
that it is finally possible to derive the indentation modulus (2) and the indentation hardness (3)
from the measurement. Figure 3.8 shows a schematic sketch of such an analysis.
Fig 3.8: a) Schematic plot of the Oliver and Pharr model b) AFM image of the
nanoindent made by a berkowich indenter
a
b

28. 26
Both values, indentation modulus as well as indentation hardness, depend strongly on the area
function A (hc) and the accuracy with which it is determined.
It is possible to improve the O&P model further by introducing a correction factor accounting
for the radial surface displacement in Sneddon's solutions (Hay et al.). In case of materials
which show a pile-up or sink-in behaviour it is furthermore possible to supplement the model
with a parameter accounting for the deviation from the ideal or calibrated area function with
respect to the real one (McElhaney et al.).
There are other models describing Dynamic contact method, CSM method, Fatigue test
method and numerous others which are a special case of the above methods.
Applications
Nanoindentation is far and wide being used in the fields of Material Science as well as
Medicine. Its applications range from measuring of basic hardness of materials to measuring
the micromechanical properties of Cobra fangs, to synthesis of nanocrystalline phases in a
glassy material.
Biomedical Applications
Applications include measurement of mechanical properties of microstructural features in
biomaterials and mapping of mechanical properties in tissues with complex microstructures.
Nanoindentation can serve as a complementary characterization tool to other techniques that
assess composition or structure with high spatial resolution, such as Raman spectroscopy,
magnetic resonance imaging, micro-computed tomography, histology, or infrared
spectroscopy. In this regard, nanoindentation has played a pivotal role in defining structure-
property relationships for tissues and their constituents.
Limitations
This tool has its own limitations, like the dependence of the results on the tip geometry and
the efficiency of the capacitors and the attenuators as well as the surface of the specimen to be
indented, which has to be smooth enough.

29. 27
3.3.2-Microhardness testing
Some of the biological samples require the determination of hardness over small areas.
Determination of hardness of individual constituents of a microstructure or checking the
hardness of a delicate sample in such samples offers a great challenge for materials scientists.
For this reason a Vickers hardness test with a square base diamond pyramid is used as a
standard for measuring the hardness of such samples. The indenter has an included angle of
1360
between the opposite faces of the pyramid. This angle was chosen because it
approximates the most desirable ratio of the indentation diameter to the ball diameter (Dieter,
1986).
Applied loads are much smaller than for Rockwell and Brinell, ranging between 1 and
1000 g. The resulting impression is observed under a microscope and measured. This
measurement is then converted into a hardness number. Careful specimen surface preparation
like grinding and polishing are necessary to ensure a well-defined indentation that may be
accurately measured. The Vickers hardness numbers are designated by HV. In general,
Vickers hardness testing is also referred to as microhardness testing on the basis of load and
indenter size. Both are well suited for measuring the hardness of small, selected specimen
regions (Callister, 1999).
Microhardness tests were performed on the same samples using a Buhler
microhardness tester attached to a light microscope equipped with a video imaging system as
shown in fig 3.9.
Fig 3.9: Photograph of vickers microhardness testing equipment

30. 28
Indents were produced using a Vickers indenter by applying a load of 10 ponds for a
period of 10 sec. Under these conditions the indents produced had diagonals between 15 and
30 µm long.
Fig 3.10: Image of a vickers microindent
The diagonal length was measured within 30 sec after the indentation, using a Buhler
hardness measuring digital eyepiece. The microhardness values were calculated using the
following equation (Dieter, 1986):
HV= 1.854 F/d2
Where HV stands for Vickers Hardness and is expressed in kg/mm2
, F is the applied force in
Newtons, and d the mean value of the diagonals in mm. The indentations were made on each
of the surfaces starting from one end of the sample to the other end.

31. 29
3.3.3- Three point bending test
A materials response to the three major forms of stress i.e. Tension, compression and shear
could be measured on a Universal testing machine commonly called as tensile testing
machine. The engineering tensile testing generally provides basic design information on the
strength of materials and is widely used as an acceptance test for the specification of
materials. The stress–strain behaviour of hard and brittle materials like shells is not usually
ascertained by a tensile test for three reasons (Callister, 1999).
• Firstly, it is difficult to prepare and test specimens having the required geometry.
• Secondly, it is difficult to grip brittle materials without fracturing them
• Thirdly, brittle materials fail after about 0.1% strain, which necessitates that tensile
specimens be perfectly aligned in order to avoid the presence of bending stresses,
which are not easily calculated.
Therefore, a more suitable transverse bending test is most frequently employed, in which a
rod specimen having either a circular or rectangular cross section is bent until fracture using a
three- or four-point loading technique. The three-point loading scheme is illustrated in Figure.
At the point of loading, the top surface of the specimen is placed in a state of compression,
whereas the bottom surface is in tension. Stress is computed from the specimen thickness, the
bending moment, and the moment of inertia of the cross section; these parameters are noted in
Figure for rectangular and circular cross sections.
Fig 3.11: A three-point loading scheme for measuring the stress–strain behavior and
flexural strength of brittle materials

32. 30
The maximum tensile stress (as determined using these stress expressions) exists at the
bottom specimen surface directly below the point of load application. Since the tensile
strengths of ceramics are about one-tenth of their compressive strengths, and since fracture
occurs on the tensile specimen face, the flexure test is a reasonable substitute for the tensile
test. The stress at fracture using this flexure test is known as the flexural strength, modulus of
rupture, fracture strength, or the bend strength, an important mechanical parameter for brittle
ceramics. For a rectangular cross section, the flexural strength
Where P is the load at fracture, L is the distance between support points, b is the breadth and d
is the depth of the sample (Callister, 1999).
For a circular cross section of radius R, the flexural strength is calculated by
Since, during bending, a specimen is subjected to both compressive and tensile stresses, the
magnitude of its flexural strength is greater than the tensile fracture strength. Furthermore, P
will depend on specimen size; with increasing specimen volume (under stress) there is an
increase in the probability of the existence of a crack-producing flaw and, consequently, a
decrease in flexural strength. Table 3.1 shows the expressions for computing stress for
rectangular and circular cross sections (Callister, 1999).
σ = Mc/I
M = maximum bending moment
c = distance from center of specimen to outer fibers
I = moment of inertia of cross section
P = applied load
Table 3.1
M c I σ
Rectangular FL/4 d/2 bd3
/12 3PL/2bd2
Circular FL/4 R ∏R4
/4 PL/∏R3
** Note: ∏ = Pi = 22/7

33. 31
The three point bending test was used to provide the values for the modulus of
elasticity in bending EB, flexural stress σf, flexural strain εf and the flexural stress-strain
response of the material. For this study the three point bend tests were carried out on two
different equipments. The preliminary tests were carried out on Zwick tensile testing machine
with a loading rate of 2 mm/sec and the second set of measurements were carried out on
indigenously constructed tensile testing equipment at a loading rate of 1 µm/ sec. The gauge
length of 15 mm was used for testing the samples in both the testing equipments.
Fig 3.12: Photograph of three point bending testing equipment
For each load- displacement curve as shown below, flexural stress, flexural strain, and
bending modulus can be calculated using the formulas.
σf = Stress at the midpoint in the material, (MPa)
εf = Strain in the material, (%)
Eb = Modulus of elasticity in bending, (MPa)
P = load at a given point on the load deflection curve, (N)
L = Support span, (mm)
b = Width of test beam, (mm)
d = Depth of tested beam, (mm)
D = maximum deflection of the center of the beam, (mm)
m = Slope of the tangent to the initial straight-line portion of the load deflection curve,
(N/mm)

34. 32
From a load displacement curve one can obtain the stress-strain curve in three point bending
by inserting the values of load and displacement in the above equations. Since, three point
bending is a pure state of bending; maximum load is concentrated always at the center. Hence
for brittle materials like shells it is sufficient to measure the dimensions of the fractured
surface by vernier callipers to calculate the values of stress and strain. In addition one can also
measure the mechanical properties as shown in the fig 3.14.
Fig 3.13: Load displacement plot of ductile material
Fig 3.14: Plot showing stress- strain behavior of ductile material and the mechanical
properties that are obtained through this diagram

35. 33
Measures of yielding (Dieter, 1986)
The stress at which the plastic deformation begins depends on the sensitivity of the strain
measurements. Various criteria for the initiation of yielding are used depending on the
sensitivity of the strain measurements and the intended use of data.
True elastic limit is calculated based on microstrain measurements at strains in the orders of
2*10-6
. This elastic limit is a very low value and is related to the motion of a few hundreds of
dislocations.
Proportional limit is calculated as the highest stress at which stress is directly proportional to
strain. It is obtained by observing the deviation from the straight line portion of the stress-
strain curve.
Elastic limit is the greatest stress the material can withstand without permanent strain and
regains its original shape on the complete release of load. With the sensitivity of strain usually
employed (10-4
), the elastic limit is greater than the proportional limit. Exact determination of
elastic limit requires a tedious loading-unloading test procedure.
Modulus of elasticity in bending
The degree to which a structure deforms or strains depends on the magnitude of an imposed
stress. For most materials that are stressed, at relatively low levels, stress and strain are
directly proportional to each other through the relationship
σ = E €
This is known as Hooke’s law, and the constant of proportionality E (GPa or psi) is the
modulus of elasticity, or Young’s modulus.
Yield strength
It is the stress required to produce a small amount of plastic deformation inside the material. It
is obtained by the stress corresponding to the intersection of the stress- strain curve and a line
parallel to the elastic part of the curve drawn from a strain of 0.2 or 0.1 percent.
Tensile Strength in bending
After yielding, the stress necessary to continue plastic deformation in materials increases to a
maximum point and then decreases to the eventual fracture. The tensile strength (MPa or psi)
is the stress at the maximum on the engineering stress–strain curve. This corresponds to the

36. 34
maximum stress that can be sustained by a structure in bending. If this stress is applied and
maintained, fracture will result.
Fracture Strength in bending
The Fracture strength (MPa or psi) is the stress at the fracture on the engineering stress–strain
curve. In the case of brittle materials like shells this value mostly corresponds to the tensile
stress as there is very less of plastic deformation inside a brittle material.
Ductility
Ductility is another important mechanical property. It is a measure of the degree of plastic
deformation that has been sustained at fracture. A material that experiences very little or no
plastic deformation upon fracture is termed brittle.
Resilience
Resilience is the capacity of a material to absorb energy when it is deformed elastically and
then, upon unloading, to have this energy recovered. The associated property is the modulus
of resilience, which is the strain energy per unit volume required to stress a material from an
unloaded state up to the point of yielding. Computationally, the modulus of resilience for a
specimen subjected to a bending test is just the area under the engineering stress–strain curve
taken up to the yield strength
Toughness
Toughness is a measure of the ability of a material to absorb energy up to fracture. Specimen
geometry as well as the manner of load application is important in toughness determinations.
For dynamic (high strain rate) loading conditions and when a notch (or point of stress
concentration) is present, notch toughness is assessed by using an impact test. Furthermore,
fracture toughness is a property indicative of a material’s resistance to fracture when a crack
is present.
For the static (low strain rate) situation, toughness may be ascertained from the results of a
tensile stress–strain test. It is the area under the stress–strain curve up to the point of fracture.
The units for toughness are the same as for resilience (i.e., energy per unit volume of
material). For a material to be tough it must display both strength and ductility and that’s the
reason behind the ductile materials which are relatively tougher than their brittle counterparts.

37. 35
3.3.4- Impact Testing
Prior to the advent of fracture mechanics as a scientific discipline, impact testing
techniques were established so as to ascertain the fracture characteristics of materials. It was
realized that the results of laboratory tensile tests could not be extrapolated to predict fracture
behavior. For example, under some circumstances normally ductile metals fracture abruptly
and with very little plastic deformation. Impact test conditions were chosen to represent those
most severe relative to the potential for fracture, namely (Dieter, 1986),
(1) deformation at a relatively low temperature,
(2) high strain rate (i.e., rate of deformation), and
(3) triaxial stress state (which may be introduced by the presence of a notch).
Fig 3.15: Schematic of loading procedure and the principle for Charpy V-notch impact
testing
Impact Testing Techniques (Callister, 1999).
Two standardized tests, Charpy and Izod, were designed and are still used to measure the
impact energy, sometimes also termed notch toughness. The Charpy V-notch (CVN)
technique is most commonly used in the United States. For both Charpy and Izod, the
specimen is in the shape of a bar of square cross section, into which a V-notch is machined.
In case of our Biocomposites the specimen is of flat rectangular crossection without the

38. 36
introduction of a notch. The load is applied as an impact blow from a weighted pendulum
hammer that is released from a cocked position at a fixed height h. The specimen is positioned
at the base as shown in fig 3.15 . Upon release, a knife edge mounted on the pendulum strikes
and fractures the specimen at the notch, which acts as a point of stress concentration for this
high velocity impact blow. The pendulum continues its swing, rising to a maximum height h*,
which is lower than h. The energy absorption, computed from the difference between h and
h*, is a measure of the impact energy. The primary difference between the Charpy and Izod
techniques lies in the manner of specimen support, as illustrated in Figure 3.15. Furthermore,
these are termed impact tests in light of the manner of load application. Variables including
specimen size and shape as well as notch configuration and depth influence the test results.
Both plane strain fracture toughness and these impact tests determine the fracture properties
of materials. The former are quantitative in nature, in that a specific property of the material is
determined (i.e., K1c ). The results of the impact tests, on the other hand, are more qualitative
and are of little use for design purposes. Impact energies are of interest mainly in a relative
sense and for making comparisons—absolute values are of little significance. Plane strain
fracture toughness tests are not as simple to perform as impact tests. Furthermore, equipment
and specimens are more expensive.
Fig 3.12: Photograph of Zwick 5102 impact testing equipment
Zwick 5102 impact tester was employed in this study to investigate the impact behavior of
these biocomposites. The compact tester is designed for determination of the impact energy
up to 5 Joules and works according to Charpy testing standards.

39. 37
3.3.5-Fracture Toughness
From fracture mechanical principles, an expression has been developed that relates
this critical stress for crack propagation (σ) to crack length (a) as ( William D. Callister )
Kc =Y* σ* √ (∏*a)
In this expression Kc is the fracture toughness, a property that is a measure of a
material’s resistance to brittle fracture when a crack is present. Kc has the unusual units of
MPa√m or psi√in. (alternatively ksi√in.). Furthermore, Y is a dimensionless parameter or
function that depends on both crack and specimen sizes and geometries, as well as the manner
of load application. Relative to this Y parameter, for planar specimens containing cracks that
are much shorter than the specimen width, Y has a value of approximately unity.
Mathematical expressions for Y have been determined for a variety of crack-specimen
geometries and these expressions are often relatively complex. For relatively thin specimens,
the value of Kc will depend on specimen thickness.
Fig 3.13: Sample preparation and loading in fracture toughness testing
However, when specimen thickness is much greater than the crack dimensions, Kc
becomes independent of thickness; under these conditions a condition of plane strain exists.
By plane strain we mean that when a load operates on a crack in the manner represented in
Figure 3.13, there is no strain component perpendicular to the front and back faces. The Kc
value for this thick-specimen situation is known as the plane strain fracture toughness K1c and
it is also defined by ( William D. Callister )
K1c=Y* σ* √ (∏*a)

40. 38
Subscript for K1c denotes that the plane strain fracture toughness for mode I crack
displacement. Brittle materials, for which appreciable plastic deformation is not possible in
front of an advancing crack, have low K1c values and are vulnerable to catastrophic failure.
On the other hand, K1c values are relatively large for ductile materials. Fracture mechanics is
especially useful in predicting catastrophic failure in materials.
The compact tension (CT) and the three point loaded bend specimen have been
standardized by ASTM (Dieter, 1986). After the notch is machined in the specimen, the
sharpest possible crack is produced at the notch root by fatiguing the specimen in a low cycle
high strain mode. The initial crack length a includes both the depth of the notch and the length
of the fatigue crack. Plain strain toughness test is generally carried out in a tensile testing
machine which provides a continous record of load P and relative displacement. A typical
load displacemnet curve for a brittle material is as shown in the figure 3.14 .The curve shows
a complete pop in instabilty where the initial crack movement continously propagates towards
failure.
Fig 3.14: Load displacement plot for a brittle material
The value of PQ determined from load displacement curve is used to calculate a conditional
value of fracture toughness denoted by KQ (Dieter, 1986).
KQ = (PQ S/BW3/2
) [ 2.9 (a/W)1/2
– 4.6(a/W)3/2
+ 21.8(a/W)5/2
– 37.6 (a/W)7/2
+ 38.7 (a/W)9/2
]

41. 39
The crack length (a) used is measured after the fracture with the help of vernier
callipers. The factor 2.5(KQ /σ0)2
is calculated and if this factor is less than both the thickness
and crack length of the specimen, then KQ is equal to K1c. Else a thicker specimen is
necessary to determine K1c.
3.3.6- Scanning Electron Microscopy
Impacted fracture surfaces, three point bending fractured samples in wet and dry
conditions and fracture toughness samples in wet and dry conditions were examined using
scanning electron microscope. Samples were cut from the fractured samples and then they
were cleaned dried and sputter coated with thinnest possible carbon coating for SEM study.
Fig 3.15: Scanning electron microscope
Scanning electron micrographs were obtained using LEO Gemini 1530 SEM equipped
with HKL technology. SEM images were generated using an accelerating voltage of 20 KV
and beam current of 3.0 nA.

42. 40
3.4-Cost effective Device for measuring mechanical properties of intricately shaped Bio-
Composites
The maximum load which a shell can withstand before it breaks depends generally on the
various physical properties of shells such as shell thickness, density, porosity, shape. Tests of
shell strength include
• Compression resistance (i.e the strength required to crush the shell between a fixed
and a moving surface )
• Puncture resistance (i.e. the force required to penetrate a shell with a small punch like
the hardness)
• Impact resistance (i.e shells resistance to force generated by a sudden impact)
Description of the tensile testing equipment
Several complex and rather expensive electronic instruments have been designed by scientists
until now to measure the strength of general engineering materials like metals, alloys,
ceramics, polymers and composites. Generally these instruments are not readily available to
biologists studying the mechanical properties of these shells. Further installing tensile testing
equipment or testing samples statistically large number of samples is relatively costly. For this
reason a simple tensile testing equipment was designed that will yield accurate data on
mechanical properties of these shells. The instrument consists of the following components.
1. Stepper motor
2. Load sensor
3. Displacement Sensor
4. Adjustable crosshead
This tensile testing machine is displacement controlled in which the operator adjusts the
displacement and the load adjusts itself to the displacement. This machine represents a screw
driven machine in which the crosshead moves at a predetermined velocity. In the case of this
tensile testing equipment the strain rate of the equipment is fixed at 1 µm/s. A constant
crosshead velocity testing machine applies a constant total strain rate that is the sum of
• elastic strain rate in the specimen
• plastic stain rate in the specimen, and
• the stain rate resulting from the elasticity of the testing machine

43. 41
Fig 3.16: Tensile testing equipment
The heavy Iron crosshead and its supports of the machine are cut from a wissenberg
camera and are used in the construction of this equipment, as it allows for a slow and smooth
increase in pressure as the crosshead is raised gradually. The crosshead of the machine moves
between two steel rods of about 12 mm in diameter and is driven over a circular screw of 15
mm in diameter. A control knob along with a butterfly nut is also provided which allows for
both coarse and fine adjustments in height. A stepper motor is attached to the moving
crosshead of the machine and the electronics of the motor are constructed such that the
crosshead moves at a strain rate of 1µm/sec. The electronic board is also constructed so that
the cross head rotates in both in clock wise and anticlockwise direction. This allows the user
to test the samples both in tension as well as compression. For high degree of accuracy in
measurements a load sensor of 5 KN from Bruster was used in the construction of this
equipment. Also a displacement sensor from Mitutyo which has accuracy of measuring 1µm
change was chosen as shown in the fig 3.16. Since the equipment works in tension and
Displacement Sensor
Load Sensor
Stepper motor
Moving crosshead
Stationary base
Control knob
Butterfly nut

44. 42
compression, different types of mechanical tests can be carried out on this equipment by
changing the grips/ supports like (Fig 3.17 a , 3.17 b)
1. Tensile testing 3. Compression testing
2. Three Point bending 4. Four point bending
This tensile testing equipment is not only used for testing the samples but also to check
the crack propagation and fracture in these materials. As such the equipment can also be
placed in the horizontal direction under a light microscope to observe the fracture.
Fig 3.17: Tensile testing machine with a) three point bending grips and b) tensile testing
grips
Since in brittle materials the crack propagation rates are faster the stepper motor is also
adjusted so that the cross head of the equipment moves at a strain rate of 1µm/sec. The
equipment also has a flexible base plate where one can insert the sample holder of their choice
to do the testing of these biological samples.
Operating the tensile testing equipment
The movable crosshead is lowered by turning the coarse control knob of the stand. As
a result, the crosshead is raised well above the shell support leaving enough space for
inserting a shell. A shell to be tested is placed in between the supports of the machine. The
a
b

45. 43
crosshead has to be lowered now and the supports should be adjusted such that the supports
just touch the shell surface. It should however be ensured that the supports do not exert any
measurable pressure on the shell by checking the reading of the load sensor. The height of the
movable crosshead is now fixed in position by a butterfly nut provided at the top of control
knob. The shell specimen is carefully positioned so that the sample is placed exactly at the
center of the supports. The butterfly nut is placed and the equipment is switched on. The
stepper motor now starts to rotate the crosshead slowly at a rate of 1µm/sec. The displacement
sensor has one of its ends fixed with the moving crosshead of the machine and the free end is
touching the base plate. If either displacement or the load is not showing zero then one can
also manually adjust both the load and displacement to zero by pressing the zero button on the
displacement sensor.
Once the shell is in position the movement of the crosshead introduces the pressure on
the shell which is transmitted through the support. The load sensor which is fixed to the
support reads the pressure measurements manually. The displacement sensor which is also
fixed to the movable crosshead shows the displacement. Following the fracture of the shell,
the system should be switched off and the crosshead has to be manually raised up to remove
the fractured pieces of the sample. The supports must be cleaned and the next set samples
could be tested in a similar procedure.
As an initial step towards the automation and efficient data collection, both the load
sensor and the displacement sensors are connected externally to a computer. The individual
load and displacement measurements are read and saved into two different excel files inside
the computer. The obtained values of load and displacement are put into one excel sheet and
they are plotted to observe the load displacement behavior of the material. From the obtained
values of load and displacement one can determine the stress- strain behavior by measuring
the sample dimensions and by substituting them in the equations of stress and strain. With the
help of stress strain graph one can determine various mechanical properties like E- modulus,
yield strength, tensile strength, ductility, resilience and toughness
This instrument can be used with minor modifications to study the fracture
propagation in shells. This type of in-situ observation can be observed by mounting the testing
equipment in the horizontal position under a light microscope. If one requires the testing of

46. 44
0.0 0.2 0.4 0.6 0.8 1.0
0
100
200
300
400
Load(N)
Displacement (mm)
Glass
Linear fit of Data for Glass
0.0 0.1 0.2 0.3 0.4 0.5 0.6
0
50
100
150
200
250
Load(N)
Displacement (mm)
Polystyrene 1
Polystyrene 2
shell as a whole without cutting into smaller fragments then one could load the whole shell
upon the three supports and could examine the mechanical properties in four point bending.
.
Fig 3.18: Load- Displacement behavior of glass in tensile testing machine in three point
bending.
Fig 3.19: Load- Displacement behavior of polystyrene in tensile testing machine in three
point bending.

47. 45
To check the effectiveness of our equipment we have calibrated by using glass as the
standard material. Since the shell specimens are mostly brittle a material which is having
almost similar characteristics like glass was chosen as a standard for calibration of the
equipment. The load-displacement plot for glass shows almost straight line behavior. In order
to verify the efficiency of the whole testing a theoretical linear fit of data was projected on the
experimental data obtained from the equipment. The plot in the fig 3.18 shows that the linear
data fit almost matches with that of the experimental fit proving that the equipment works fine
with brittle materials like shells. For checking the consistency of the results produced by the
equipment two polystyrene samples of similar dimensions were tested in three point bending.
From the results shown in the fig 3.19, it is clear that the results obtained from the equipment
are consistent and very much reliable.

48. 46
2.0 2.5 3.0 3.5
0
20
40
60
80
100
E-Modulus(GPa)
Hardness (GPa)
Dry Condition
Wet Condition
Chapter-4
Results and Discussions
4.1 Results
Illustrative indentation plots for Shell samples of Gari stangari and Elliptio crassidens
in dry and wet are shown in figure 4.1 and figure 4.2. Significant differences were observed in
mechanical properties during the hold periods on both the wet samples.
Fig 4.1: Variation of E- modulus and hardness in dry and wet conditions for the Gari
stangari.
The appreciable changes in the mechanical properties of these samples according to the
position of the indent indicate that these shells have anisotropic behaviour. Hardness and
elastic modulus were determined from unloading curves using the procedure of Oliver and
Pharr. Although the reliability of absolute values has been questionable due to the accuracy in
determining the contact depth (and hence area) on anisotropic materials, the unloading slope
approach should be sufficient to determine the relative changes which we are concerned with
here. Testing wet shows some difference between the two materials with greater decreases in
hardness and modulus for Gari stangari than Elliptio crassidens, particularly, for the same
indentation depths. The data implies that Gari stangari is more effectively plasticized than
Elliptio and water is more effective in plasticizing the surface layers of Gari stangari.

49. 47
2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8
50
55
60
65
70
75
80
85
90
E-Modulus(GPa)
Hardness(GPa)
Dry Condition
Wet Condition
Fig 4.2: Variation of E- modulus and hardness in dry and wet conditions for Elliptio.
Fig 4.1 and fig 4.2 shows how the hardness and reduced modulus vary with position of the
indent and test medium. Hardness and elastic modulus determined from the Oliver and Pharr
procedure are almost invariant with load, increasing by ~10% for the lowest indentation load
compared with the largest.
In case of Gari stangari, the average hardness in dry condition was 3.14 +/- 0.48 GPa and in
wet condition it was found out to be 1.97 +/- 0.32 GPa. The average E- Modulus in dry
condition was 73.60 +/- 11 GPa and in wet condition was 33.8 +/- 3.7 GPa. The change is
very significant and it accounts about 37% change in hardness and about 54.1% change in E-
Modulus between the wet and dry conditions and is shown in the Fig 4.1. The ratio of
Hardness to E- Modulus was found out to be 0.043 in dry condition and is increased to 0.058
in wet conditions.
For Elliptio crassidens, the average hardness in dry condition was 3.15 +/- 0.35 and in wet
condition it was found out to be 2.69 +/- 0.25. The average E- Modulus in dry condition was
71 +/- 9 GPa and in wet condition was 54 +/- 3.9. The change here also is significant and it
accounts about 15% change in hardness and about 24.1% change in E-Modulus between the
wet and dry conditions as shown in the Fig 4.2. The ratio of Hardness to E- Modulus was
found out to be 0.044in dry condition and is increased to 0.05 in wet conditions.

50. 48
3.6 4.0 4.4 4.8
60
64
68
72
76
80
E-Modulus(GPa)
Hardness (GPa)
Dry Condition
Wet Condition
2.8 3.2 3.6 4.0 4.4 4.8
60
70
80
90
100
110
E-Modulus(GPa)
Hardness (GPa)
Dry Condition
Wet Condition
Fig 4.3: Variation of E- Modulus and hardness in dry and wet conditions for Dosina
anus in the first measurement.
Fig 4.4: Variation of E- modulus and hardness in dry and wet conditions for Dosina
anus in the second measurement.

51. 49
In case of Dosina anus, the Average hardness in dry condition was 3.77 +/- 0.5 GPa
and in wet condition it was found out to be 3.6 +/- 0.56 GPa. The average E- Modulus in dry
condition was 72.3 +/- 5 GPa and in wet condition was 88.5 +/- 14 GPa. The ratio of
Hardness to E- Modulus was found out to be 0.052 in dry condition and is decreased to 0.042
in wet conditions. There is a slight reduction in hardness of 3.2% but at the same time there is
a significant increase in the E-modulus or the stiffness of 22.4% the sample as shown in Fig
4.3. This increase in E-Modulus is rarely seen in general engineering materials. Also in
biomaterials like bones, teeth and nacre there will be a decrease in E- Modulus when they are
stored in water.
To confirm this peculiar behaviour, and to ensure that the difference in E- Modulus
obtained by the nanoindentor is not because of the anisotropy, the same shell was tested again
in dry and wet conditions. For statistical support, the samples were subjected to 15 indents in
dry and 15 indents in wet conditions. In the second attempt too there was a significant
increase in the E- Modulus as shown in Fig 4.4. The Average hardness in dry condition was
4.2 +/- 0.35 GPa and in wet condition it was found out to be 3.77 +/- 0.3 GPa. The average E-
Modulus in dry condition was 62.5 +/- 2.7 GPa and in wet condition was 75 +/- 3.78 GPa.
There is a slight reduction in hardness of 11.7% and again there is a significant increase in the
E-modulus of 20% between the wet and dry conditions. The ratio of Hardness to E- Modulus
was found out to be 0.067 in dry condition and is increased to 0.05 in wet conditions. The
differences between the mechanical properties in dry and wet conditions of Gari stangari,
Elliptio Crassidens and Dosina anus are as shown in Table 1. From the table it is clear that
the hardness is lower in wet state compared to dry state in all the samples. It is also clear that
the shells have lower modulus in wet conditions except in the case of Dosina anus. Also the
Hardness to modulus ratio shows an increase in the case of Gari stangari and Eliiptio
crassidens from dry to wet condition, but it shows a decrease in case of Dosina anus.
The indentation experiments on shells in wet condition were performed in air
immediately after rewetting the samples that were stored under tap water for 60 hrs. There
were 10 to 20 indents taken for the comparison purpose and the total time for the indentation
process varied from two to 3 hours. The equipment took approximately 30 minutes for drift
correction and time for each indentation was approximately 10 minutes. The results from the
previous studies on human teeth enamel show a significant change in elastic modulus and
hardness between dried state and the samples constantly stored and investigated under a fluid
(Guidoni et al., 2006). If the wet sample is taken out of the fluid and within a few minutes is

52. 50
tested by indentation, a transition from totally wet state to fairly moist state occurs. Both the
hardness and E-modulus increase and reach more or less steady state within 10 min (Schöberl
et al., 2006).
Table 1: anoindentation values in dry and wet conditions for Gari stangari, Elliptio
crassidens and Dosina anus
Sample Avg Max
Depth (nm)
Avg Max
load
(m )
Avg
Hardness
(GPa)
Avg E-
Modulus
(GPa)
Hardness/E-
modulus
ratio
Gari stangari
(Dry)
528 18 3.13 +/- 0.48 73.6 +/- 11 0.043
Gari stangari
(Wet)
531 10 1.98 +/- 0.32 33.8 +/- 4 0.058
Elliptio
(Dry)
518 17 3.15 +/- 0.35 71.1 +/- 9 0.044
Elliptio
(Wet)
535 15 2.7 +/- 0.25 54 +/- 4 0.050
Dosina anus 1
(Dry)
505 19 3.77 +/- 0.5 72.3 +/- 5 0.052
Dosina anus 1
(Wet)
546 22 3.65 +/- 0.56 88.6 +/- 15 0.042
Dosina anus 2
(Dry)
545 23 4.20 +/- 0.35 62.5 +/- 3 0.067
Dosina anus 2
(Wet)
563 23 3.77 +/- 0.3 75 +/- 4 0.050
It is reasonable to assume that 24 hours immersion is more than adequate for
saturation of the surface layers. However, as these samples are biomaterials they are wetted
for 3 days before testing. They were few questions about the reliability of such a testing
procedure. Since the samples were removed from the water and were tested in air and also as
the indenter takes about 30 minutes for the drift correction, there were doubts whether the
sample had already reached the dry condition during the indentation process. For this reason
weight loss measurements were carried out on these samples.
For the weight loss measurements shell specimens were polished to similar sizes of
that of standard specimens. In our case the standards like austenitic stainless steel and glass
were used. The polished shell samples and standards were both weighed in dry condition.
Then they were immersed in water for three days and the samples were once again weighed in
regular intervals of time and the weights were noted as a function of time.

53. 51
0 60 120 180
0.4625
0.4630
0.4635
0.4640
0.4645
Weight(gm)
Time (min)
Elliptio Wet
Elliptio Dry
0 5 10 15 20 25
1.3525
1.3530
1.3535
1.3540
1.3545
Weight(gm)
Time (min)
Austinitic Stainless Steel Wet
Austinitic Stainless Steel Dry
Fig 4.5: Variation of weight as a function of time in dry and wet conditions for Elliptio
Crassidens.
Fig 4.6: Variation of weight as a function of time in dry and wet conditions for
austenitic stainless steel.
0 2000 4000 6000 8000 10000
0.4625
0.4630
0.4635
0.4640
0.4645
Weight(gm)
Time (min)
Elliptio Wet
Elliptio Dry

54. 52
0 2 4 6 8 10 12 14 16
0.3035
0.3040
0.3045
0.3050
0.3055
Weight(gm)
Time (min)
Glass Dry
Glass Wet
Fig 4.7: Variation of weight as a function of time in Dry and Wet conditions for Dosina
anus.
Fig 4.8: Variation of weight as a function of time in Dry and Wet conditions for Glass.
0 1500 3000 4500 6000
1.312
1.316
1.320
1.324
Weight(gm)
Time (min)
Donina anus Dry
Dosina anus Wet

55. 53
0.000 0.005 0.010 0.015 0.020 0.025 0.030
0
10
20
30
40
50
Stress(MPa)
Strain
Reference 1
Reference 2
Reference 3
From the weight loss measurements it is clear that the phenomenon is not only taking
place at the surface but the phenomenon is also predominantly taking place at the bulk level.
Also we could see that, it takes approximately 3 hours to evaporate half of the water from the
shells as shown in fig 4.5 and 4.7. This proves that the shells have still retained half of the
water in them even after the nanoindentation measurements and the process of testing them in
air in nanoindentation apparatus most likely represents the in vivo testing of the samples.
Further after observing that the phenomenon is predominantly happening in the bulk of the
sample, additional research was focused on the influence of wetting on the mechanical
properties at the macroscopic level by using classical mechanical tests like the three point
bend tests, impact tests and fracture toughness tests.
Fig 4.9: Variation of E- modulus, fracture strength and toughness for a reference
sample3
of Gari stangari in dry conditions.
In the macroscopic bend tests, initially a reference sample1
of Gari stangari was tested
for the purpose of comparison as shown in the fig 4.9. As explained previously in the
specimen preparation for bending tests, the samples were cut from one particular shell with
their edges being ground and polished and they were numbered in ascending order from 1, 2
and 3 according to their position from left to right. From the stress- strain plot shown in the
3
Note : Here the reference sample refers to the sample which is in dry sate

56. 54
fig 4.10, it is clear that these samples are stronger and tougher at the anterior end and their
strength and toughness decreases gradually towards the posterior end of the shell.
Fig 4.10: Variation of E- modulus, fracture strength and toughness in dry and wet
conditions for Gari stangari.
Since we see from the above results of the reference samples that the mechanical
properties of these materials depend upon the position, samples for the comparison tests were
cut very close to one another. The whole shell was cut into four parts and the samples from
alternate positions were soaked in tap water for 60 hrs (approx 3 days) before testing. The
samples were then tested in three point bending at a strain rate of 2 mm/sec. From the results
we see that the samples in wet condition show higher stresses and higher strains compared to
the ones in dry condition. Although there is not much of change in the elastic modulus
between the dry and wet samples, there is a significant change in tensile strength, ductility and
toughness in the hydrated samples.
Also one could see a common difference in the stress-strain behavior in wet and dry
samples. It is seen that in these biocomposites, there are saw tooth-like stress strain curves.
Even though there is a crack initiation inside the wet sample the sample is able to take higher
and higher load before it surrenders itself to fracture. Although we see a change in mechanical
0.00 0.01 0.02 0.03 0.04
0
20
40
60
80
B1- Wet
B2- Dry
B3- Wet
B4- Dry
Stress(MPa)
Strain

57. 55
0.00 0.02 0.04 0.06 0.08 0.10
0
10
20
30
40
Stress(MPa)
Strain
Reference 1
Reference 2
Reference 3
Reference 4
Reference 5
Reference 6
properties between the wet and dry samples, it is difficult to comment that the change is
entirely because of hydration. For this reason, tests were carried out on another species of
mollusc namely Dosina anus.
Fig 4.11: Variation of E- modulus, fracture strength and toughness for a reference
sample of Dosina anus in dry conditions.
Initially a reference sample of Dosina anus was tested for the purpose of comparison
of mechanical properties in dry conditions. As explained previously, the samples were cut
from one particular shell with their edges being ground and polished and they were numbered
in ascending order from 1, 2, 3, 4, 5 and 6 according to their position from anterior to
posterior end. From the stress- strain plot it is clear that these samples are having almost the
same strength and toughness in dry conditions (Fig 4.11). Since we see from the above results
of the reference sample that the mechanical properties of these shell materials are almost
independent of the position, the samples for the comparison tests between wet and dry
conditions were cut very close to one another. The whole shell was cut into seven parts and
the samples from alternate positions were soaked in tap water for 60 hrs (approx 3 days)
before testing. The samples were then tested in three point bending at a strain rate of 2
mm/sec. From the results we see that the samples in wet condition show higher stresses and
higher strains compared to the ones in dry condition as shown in fig 4.12. Although there is a
slight decrease in the elastic modulus between the dry and wet samples, there is a significant

58. 56
change in tensile strength, ductility and toughness in the hydrated samples. The wet samples
show almost a two fold increase in strength. Further the ductility of the sample is also
enhanced by wetting. Additionally, even the toughness is increased because of the increase in
both strength and ductility in wet conditions. With an exception of sample C5 all the rest of
the samples follow the same trend.
Fig 4.12: Variation of E- modulus, fracture strength and toughness in dry and wet
conditions for Dosina anus.
The fractured surfaces of the samples were observed with SEM. The fracture observed
in both the dry and wet condition is of a mixed type which is neither entirely ductile nor
entirely brittle. Also, from the fractured surfaces shown in fig 4.13 and fig 4.14, it is evident
that, in wet condition, more organic matrix is available for deformation, producing more
fibrils. The dehydration leads to less plastic deformation of the matrix and less energy
dissipation. It is seen clearly that larger matrix fibrils were pulled out from the wet sample as
compared with dry samples, which indicates more plastic deformation of organics during the
bend testing. SEM images in fig 4.15 and fig 4.16 at a higher magnification show that some
were still wrapped inside organic matrix, showing the stronger interfacial bonding between
carbonate particles and organic matrix in hydrated shells.
0.00 0.04 0.08 0.12 0.16 0.20
0
10
20
30
40
50
60
C1- Wet
C2- Dry
C3- Wet
C4- Dry
C5- Wet
C6- Dry
C7- Wet
Stress(MPa)
Strain

59. 57
Fig 4.13: SEM image of fractured surface of Dosina anus tested in wet condition during
the bending test.
Fig 4.14: SEM image of fractured surface of Dosina anus tested in dry condition during
the bending test
Larger
matrix fibrils

60. 58
Fig 4.15: SEM image of fractured surface of Dosina anus tested in wet condition during
the bending test.
Fig 4.16: SEM image of fractured surface of Dosina anus tested in dry condition during
the bending test.
Carbonate
particles
wrapped inside
the matrix

61. 59
All the three point bending tests so far were carried out on a Zwick tensile testing
machine which operates at a strain rate of 2 mm/sec. Since there are only few shells which are
tested for observing this phenomenon, few more tests were carried out on Gari stangari and
Dosina anus in wet and dry conditions. In order to test the efficiency and effectiveness of the
cost effective tensile testing equipment and to confirm the phenomenon taking place in these
mollusc shells, supplementary tests were carried out for statistical support. As said previously
the tensile testing equipment which is built indigenously has a strain rate of 1 µm/sec. Hence
we observe a change in the load- displacement behaviour compared to the previous plots.
Initially for the four point bend test, two shells of similar sizes and shapes are chosen. One of
the samples was put in water for three days and was tested along with the other dry sample.
Fig 4.17: Variation of E- modulus, fracture strength and toughness in dry and wet
conditions for Dosina anus in four point bending by indigenously built tensile testing
machine.
From the results obtained from the equipment ( fig 4.17) it is clear that the shells are
gaining their strength and toughness in water in the longitudinal direction. It is worth while to
compare the shells in two conditions without normalizing the plots to stress and strain as the
shells are of similar size and shape. There is about 23% increase in strength and 26% increase
in ductility in wet conditions.
0.00 0.04 0.08 0.12 0.16 0.20
0
100
200
300
400
Load(N)
Displacement (mm)
Dry Condition
Wet Condition

62. 60
0.00 0.02 0.04
0
20
40
60
Load(N)
Displacement (mm)
Dry Sample
Wet Sample
The second set of tests was carried out on Gari stangari with the samples being
polished before to similar sizes. One of the samples was kept in water for 3 days and was
tested along with the dry sample in three point bending.
Fig 4.18: Variation of E- modulus, fracture strength and toughness in dry and wet
conditions for Gari stangari in 3 point bending by indigenously built tensile testing
machine.
Also three point bending results on Gari stangari show that these shells are gaining
mechanical properties in hydrated conditions (Fig 4.18). There is about 23% increase in
strength and 12% increase in ductility in wet conditions. From the above two results the E-
modulus in Dosina anus seems to be lesser in wet conditions compared to that of dry
conditions. Further, the E- Modulus in Gari stangari is relatively higher in wet conditions
compared to the dry conditions. These results contradict the results obtained from the
nanoindentation tests. One of the main reasons for this sort of a behavior should be attributed
to the structure. Since these shells have hierarchical structure, the structure in the longitudinal
direction differs significantly from the structure in transverse direction. It is worth to note in
this aspect that the three point bend tests are carried out in the longitudinal direction and the
nanoindentation tests are carried out in vertical directions of the shell.

63. 61
0.0 0.2 0.4 0.6 0.8
0
5
10
15
20
25
Stress(MPa)
Strain (%)
Dry Condition
Wet Condition
In order to confirm that the mechanical properties depend upon the loading direction
two pieces of shell of similar sizes and shapes of Elliptio crassidens were tested in transverse
direction in three point bending.
Fig 4.19: Variation of E- modulus, fracture strength and toughness in dry and wet
conditions for Elliptio in 3 point bending in transverse direction by indigenously built
tensile testing machine.
Also from figure 4.19 one could observe that there is a change in E- Modulus in the
macroscopic bend tests which are performed on the samples in transverse direction. These
shells show an increased ductility of 15 % and 30 % decrease in fracture strength and
toughness when they are stored in water. Also there is a 35% decrease in the E- Modulus
which approximately coincides with the decrease in E- Modulus seen through nanoindentation
testing.
The above sets of tests were carried out in tap water. In view of the fact that both Gari
stangari and Dosina anus are sea water molluscs instead of fresh water species, three point
tests were carried out by immersing the samples in sea water for a period of three days. The
samples preparation and procedures are similar to the tests explained previously except that
the samples are stored in sea water. From the results in fig 4.20, 4.21, 4.22, 4.23, apart from
the sample A8, it is obvious that the shells are gaining their mechanical properties even in sea

64. 62
water. Although the increase in mechanical properties is not that significant as fresh water,
still these results set the trend for a similar phenomenon that is taking place inside these
shells.
Fig 4.20: Variation of E- modulus, fracture strength and toughness in dry and wet (sea
water) conditions for Dosina anus in 3 point bending.
Fig 4.21: Variation of E- modulus, fracture strength and toughness in dry and wet (sea
water) conditions for Dosina anus in 3 point bending.
0.00 0.01 0.02 0.03 0.04
0
10
20
30
40
Stress(MPa)
Strain
A1 Wet
A2 Dry
A3 Wet
A4 Dry
A5 Wet
0.00 0.01 0.02 0.03 0.04
0
10
20
30
40
50
60
70
Stress(MPa)
Strain
A5 Wet
A6 Dry
A7 Wet
A8 Dry
A9 Wet

65. 63
Fig 4.22: Variation of E- modulus, fracture strength and toughness in dry and wet (sea
water) conditions for Dosina anus in 3 point bending.
Fig 4.23: Variation of E- modulus, fracture strength and toughness in dry and wet (Sea
water) conditions for Dosina anus in 3 point bending.
0.00 0.01 0.02 0.03 0.04
0
10
20
30
40
Stress(MPa)
Strain
B1 Dry
B2 Wet
B3 Dry
B4 Wet
B5 Dry
0.00 0.01 0.02 0.03 0.04
0
20
40
60
80
Stress(MPa)
Strain
B5 Dry
B6 Wet
B7 Dry
B8 Wet
B9 Dry

66. 64
1 2 3 4
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Impacttoughness(J/mm
2
)
Sample position
Elliptio Polished
Elliptio Unpolished
In order to support the results obtained from the three point bend tests and to confirm
that there is an increase in toughness, impact tests were carried out on wet and dry samples of
Elliptio crassidens. Initially to test whether there is any influence of sample preparation and
polishing on the impact properties of shell materials, few tests were carried out between
polished and unpolished samples. The unpolished samples are not only difficult to load inside
the impact testing machine but also produces results which are inconsistent as shown in the
fig 4.24. In addition, the area determination for an unpolished specimen is relatively difficult
and laborious process.
Fig 4.24: Variation of impact toughness between polished and unpolished samples of
Elliptio
For this reason the samples are finely polished before the testing process. Since
Elliptio is a fresh water mollusc the samples are stored in tap water for 3 days before testing
them. In general, Impact testing provides toughness qualitatively. Therefore, couple of
samples are tested simultaneously to get an average value of fracture toughness as shown in
fig 4.25. From the results obtained through impact tests the average impact toughness was
calculated for hydrated and dehydrated samples. The samples which were stored in water had
an average impact toughness of 2.72 J/mm2
compared to the dry samples which showed an
average impact toughness of 2.37 J/mm2
(Fig 4.26). The results clearly show that the samples

67. 65
which were stored in water have higher toughness of approximately 15% compared to the
dehydrated samples.
Fig 4.25: Influence of water on impact toughness of samples of Elliptio
Fig 4.26: Averaged impact toughness for samples of Elliptio in dry and wet conditions
0 1 2 3
0.0
0.5
1.0
1.5
2.0
2.5
AverageImpactToughness(KJ/mm
2
)
Sample Condition
Dry Condition
Wet Condition