i

ANALYSIS OF THE CALCIUM CONTENT AND DILUENT PROPERTIES OF

MILKFISH (Chanos chanos) AND MANGROVE JACK BONES (Lutjanus
argentimaculatus)

A Research Paper
Presented to
The Faculty of Philippine Science High School Western Visayas
Bito-on, Jaro, Iloilo City

In Partial Fulfilment
of the requirements for
SCIENCE RESEARCH II

BY

Joseph Benedict Arenas

Joseph Jeffrey Garcia
Ruzzel Ragas
Fourth Year Tau

January 2011

Arenas, Joseph Benedict B., Garcia, Joseph Jeffrey A., Ragas, Ruzzel D. Analysis of the
calcium content and diluent properties of milkfish (Chanos chanos) and mangrove jack bones
(Lutjanus argentimaculatus). Unpublished Research. Philippine Science High School Western
Visayas Campus, Bitoon, Jaro, Iloilo City. February 2010.
Abstract
This study aimed to measure the calcium carbonate content and the physical properties of
milkfishs and mangrove jacks bones to be used as diluents in terms of particle size, particle
shape, powder flow, moisture content, bulk density and tapped density. It also aimed to compare
the physical properties of milkfishs and mangrove jacks bones as diluents with the
pharmaceutical standard. Using physical analysis of diluent properties and calcium carbonate
assay we determined that the calcium carbonate content of the mangrove jack bone samples and
milkfish bone samples are 45.12% and 41.78% respectively and its physical properties did not
pass the standards and is not recommendable.

TABLE OF CONTENTS
Abstract
List of Tables
List of Plates
List of Appendices
Chapter
I. INTRODUCTION
A. Background
B. Statement of the Problem
C. Objectives
D. Experimental Design
E. Significance of the Study
F. Scope and Delimitation
G. Definition of Terms
II. REVIEW OF RELATED LITERATURE
A. Milkfish
B. Mangrove Jack
C. Diluent
D. Calcium carbonate
E. Related Studies
III. METHODOLOGY
A. List of Materials and Equipment
B. Procurement of Chemicals and Materials
C. Collection of Milkfish and Mangrove jack Samples
D. Preparation of Fish Bone Samples
E. Chemical Testing of Fish Bone Samples
F. Analysis of Calcium Carbonate Physical Properties
G. Disposal of Chemical and Biological Waste
IV. RESULTS AND DISCUSSION
A. Results
B. Discussion
V. CONCLUSION AND RECCOMENDATION
A. Summary of results
B. Conclusion
C. Recommendation
Literature cited
Appendices

PAGE
i
ii
iii
iv

1
2
3

LIST OF TABLES
Table

Page

1. Interpretation of angles of repose

2. Interpretation of Hausner Ratios
3. Interpretation of particle shape
4. Interpretation of particle size
5. Interpretation of moisture content
6. Calcium carbonate content and physical properties of milkfish
and mangrove jack bones as diluent

LIST OF PLATES
Plates

Page

1. Mangrove jack particle shape (trial 1)

2. Mangrove jack particle shape (trial 2)
3. Mangrove jack particle shape (trial 3)
4. Milkfish particle shape (trial 1)
5. Milkfish particle shape (trial 2)
6. Milkfish particle shape (trial 3)
7. Sieving of milkfish sample
8. Calcium carbonate assay of mangrove jack sample
9. Weighing of milkfish sample for moisture content
10. Preparation of powder flow funnel setup
11. Measuring of volume for tapped density of mangrove jack sample

LIST OF APPENDICES
APPENDIX
A. Raw Data
B. Photos

CHAPTER 1
INTRODUCTION
A. Background of the Study

Diluents or fillers are additives that create in bulk thus, making a material larger
without affecting its nutritional value. They increase the volume of materials by filling
the empty spaces. Diluents or fillers are commonly used in making medicinal dosage
forms have bigger bulk. Diluents can help the dosage forms break apart once it is in the
digestive system. Common diluents are lactose, microcrystalline cellulose, starch,
powdered sucrose and calcium phosphate (Ansel and others 1999).
One of the few common sources of diluents is calcium carbonate. Calcium
carbonate is a chemical compound that is derived from calcium and is present in bones.
Oyster, dolomite and bone meal are common sources of calcium carbonate and are used
as calcium supplements because these ingredients meet the minimum calcium content but
the lead content in most oyster shell and dolomite surpass the U.S.A. lead federal limit;
however most bone meal sources meet the U.S.A lead federal limit. It is established that
the bones of fish contains calcium carbonate and that the bones have low amount of lead
(Scelfo and Flagel 2000. Bones of other fishes such as the giant seaperch (Lates
calcarifer Bloch) and hoki (Macruronus novaezelandiae) have been established as a
possible source for calcium carbonate (Phiraphniyo and others 2006). ). Milkfish and
mangrove jack bones may be a potential source of calcium because it is described as
bony fishes.
Milkfish and mangrove jack are common fishes sold in markets of Southeast Asia
which are of high demand (SEAFDEC 2009).
Milkfish (Chanos chanos) is the only living species in the family Chanidae; it is
an important source of food in the coastal regions of South Asia like in the Philippines.
Milkfish only spawn in completely brackish waters. Milkfish is one of the most
commonly seen fish in Philippine markets. Boneless milkfish is common food for
foreigners and native Filipinos and only the meat is eaten. These fishes are subjected to
the deboning industry and are processed into boneless fish meals because of their bony
flesh. The bones that are deboned by these industries are disposed as trash (Bagarinao
1999).
Mangrove jack (Lutjanus argentimaculatus) is a vital food fish, it has a high
market price and of high demand in Southeast Asia. Mangrove jack is commonly found

in mangrove-lined estuarine systems. It migrates to the offshore reefs to reproduce. It is

also known as a powerful sport fish (Fishbase 2010).
Milkfishs and mangrove jacks bones have the potential to contain calcium
carbonate and calcium carbonate is established to be a common source of diluents.
Milkfishs and mangrove jacks bones are probable sources of diluents.
This study will determine if the physical properties of the bones of milkfish and
mangrove jack can be utilized to be a source of diluents.
B. Statement of the Problem
Evaluate the calcium carbonate content and physical properties of the bones of
milkfish and mangrove jack as standard diluents?
C. Objective
This study aims to:
1.) Measure the calcium carbonate content of milkfishs and mangrove jacks bones.
2.) Measure the physical properties of milkfishs and mangrove jacks bones to be used as
diluents, in terms of:
a) particle size
b) particle shape
c) powder flow
d) moisture content
e) bulk density

Particle Size

f) tapped density

3.) Compare the physical properties of milkfishs and mangrove jacks bones as diluents
Particle Shape
with
the pharmaceutical
standard.
Milkfish
Bone
Diluent
jack Design
D.Mangrove
Experimental
Bones

Moisture Content
Bulk Density
Tapped Density
Powder Flow

Calcium
carbonate content

E. Significance of the Study

Most studies on fishes are focused mostly on the breeding of fishes and the
quality of the meat. The results of this study can add information on the utilization of the
bones of milkfish and mangrove jack.
If successful, the results of this study may be beneficial to both the
pharmaceutical and fish-breeding industries by making the usually disposed fish bones
useful by utilizing it as diluents. This may also provide a probable cheap alternative for
diluents as opposed to the common sources.
This study may also lead to the development of studies relating to tablet
formulation and the possibility of using fish samples as a source of active ingredients.
F. Scope and Delimitations
This study only dealt with the possibility of the bones of milkfish (Chanos chanos)
and mangrove jack (Lutjanus argentimaculatus) to be used as diluents and not any of the
other pharmaceutical uses.
The method that was used in determining the calcium carbonate content was the
calcium carbonate assay derived from USP 2007. Calcium carbonate that was measured
will be the amount from the bones of the milkfish and mangrove jack. All other parts
which may or may not contain calcium will not be measured.

The results of the study only concerned the physical characteristics of the
powdered bones as a diluent. The properties to be tested will only include particle size,
particle shape, powder flow, moisture content, bulk density and tapped density.
G. Definition of Terms
Bulk density- Bulk density is considered as the loose density of a powder because it is
measured without compacting the powder. Bulk density is expressed as mass of the
powder per unit volume (GEA 2010).
Calcium Carbonate Calcium carbonate is a chemical compound. The chemical
formula CaCO3 covers a raw material, which is widespread throughout nature, whether
dissolved in rivers and oceans, in molten form as cold carbonatite-lava, or solid as a
mineral in the form of stalactites, stalagmites or as the major constituent of whole
mountain ranges. Plants and animals need calcium carbonate to form their skeletons and
shells (IMA 2009).
Diluents- Diluents are inert substances used as fillers to create the desired bulk, flow
properties, and compression characteristics in the preparation of tablets and capsules
(Pharmpedia 2008).
Mangrove jack (Lutjanus argentimaculatus) Mangrove jack is a powerful sport fish
that has excellent eating that are commonly found in mangrove-lined estuarine
(Fishbase 2010).
Moisture content- Moisture content is the amount of liquid in a certain substance and
can be determined by loss on drying which is a procedure that determines the amount of
volatile matter of any kind that is driven off under the conditions specified (USP-NF
2007).
Milkfish (Chanos chanos) Milfkish is the sole species in the Family Chanidae in the
Order Gonorynchiformes and is most closely related to the freshwater Ostariophysi.

Milkfish have matured and spawned under various conditions of captivity, and hatcheries
have produced larvae to supply the culture ponds (Bagarinao 1999).
Particle size- Particle size is the mean diameter of one particle (Brittain 2002).
Particle shape- Particle shapes of each particle that can be defined in terms of
elongation, convexity and circularity. Commonly used shapes to define particles are
acicular, columnar, flake, plate, lath and equant (USP-NF 2007).
Powder flow- Powder flow is the behavior of a certain powder or is the ability of a
powder to flow. Powder flow is usually a considered as a one dimensional characteristic
of powders (Prescott and others 2000).
Tapped density- Tapped density is considered as the compact density of a powder
because it is measured by tapping the powder also expressed as mass of the powder per
unit volume (Thomas 2010).

CHAPTER 2
REVIEW OF RELATED LITERATURE

1. Fishes
1.1. Milkfish (Chanos chanos)
Milkfish was described by the scientific community by Petrus Forsskal in 1775. It
is the sole species in the Family Chanidae in the Order Gonorhynchiformes. It was first
described as Mugil chanos by Petrus Forsskal in 1775 based on a juvenile specimen from
the Red Sea. Lacepede used the name Chanos arabicus in 1803, and Klunzinger used the
valid name Chanos chanos in 1871. Milkfish (Chanos chanos) and milkfish (Chanos
chanos) farming are very important to the diet and economy of the people of the
Philippines. Iloilo is one of the top producers of milkfish in the Philippines (Bagarinao
1999).
Adults occur in small to large schools near the coasts or around islands where
reefs are well developed. Eggs and larvae are pelagic up to 2-3 weeks. Older larvae
migrate onshore and settle in coastal wetlands (mangroves, estuaries) during the juvenile
stage, or occasionally enter freshwater lakes. Juveniles and sub-adults return to the sea
where they mature sexually. Spawn only in fully saline water. Larvae eat zooplankton;
juveniles and adults eat cyanobacteria, soft algae, small benthic invertebrates, and even
pelagic fish eggs and larvae. Larvae are collected from rivers and are grown in culture
ponds into juveniles, which are marketed fresh, smoked, canned and frozen. Brood stocks
can be raised and spawned in captivity to produce larvae in the hatchery (Fishbase 2010).
Milkfish (Chanos chanos) has approximately 38 branching spines along the
ventral muscles. The bones of the milkfish (Chanos chanos) compose approximately
22%-25% of the weight of the whole fish. (Bagarinao 1999)
Milkfish is established by the USDA National Nutrient Database for Standard
Reference to have 55 mg of calcium per 100 g serving (USDA 2007).

1.2. Mangrove Jack (Lutjanus argentimaculatus)

The Mangrove Jack (Lutjanus argentimaculatus) is greenish brown to reddish.

Juveniles have pale bars on the sides of the body and one or two blue lines on the cheeks.
The species has an emarginated caudal fin and scale rows on the back that are roughly
parallel to the lateral line. Juveniles and young adults occur in mangrove estuaries, the
lower reaches of freshwater streams and tidal creeks. Adults are often found in groups
around coral reefs. Eventually migrate offshore to deeper reef areas, sometimes
penetrating to depths in excess of 100 m. Mangrove Jack are mainly nocturnal, this
species feeds mostly on fishes and crustaceans.

Mangrove Jack (Lutjanus

argentimaculatus) is an important market species throughout the Indo-Pacific region, but

never found in large quantities. Mangrove Jack is a good aquaculture species because it
doesnt get rancid easily when frozen. It commands a good export market price with no
limit on body size. Max length is 104 cm, max weight 14.5 kg and max age 39 years for
specimens from the east coast of Australia (Fishbase 2010).
2. Calcium Carbonate
2.1. General information
Bone generally is composed of 2/3 mostly calcium, phosphate and carbonate the
rest is organic material (WBI 2000).
Calcium Carbonate is an exceptional mineral. The chemical formula CaCO3
covers a raw material, which is widespread throughout nature, whether dissolved in rivers
and oceans, in molten form as cold carbonatite-lava, or solid as a mineral in the form of
stalactites, stalagmites or as the major constituent of whole mountain ranges. Plants and
animals need calcium carbonate to form their skeletons and shells. In fact, when
considering our lives, modern mankind could hardly imagine existing without calcium
carbonate. Almost every product in our daily lives either contains calcium carbonate or
has some association with the mineral during its production (IMA 2009).
Calcium carbonates common properties are: it absorbs moisture and is hardly
dissolved in water. It is not soluble in alcohol, but a bit dissolved in the water with
ammonium salt or carbon dioxide in it. It effervesces and will be dissolved as soon as it
touches dilute acetic acid, hydrochloric acid and nitric acid (SYNCL 2008).

Calcium carbonate is usually used as co-processed diluents and is manufactured

with a mixture of other substances; one name brand is Calcium 90 which composes 90%
calcium carbonate and 9% starch (Pharmpedia 2005).
2.2. Method of determining calcium carbonate
Calcium carbonate content is determined through calcium assay as described in
the USP. 200 mg of bone sample which was previously dried at 200C for four hours will
be transferred to a 250 mL beaker. The sample will be moistened thoroughly with about 3
mL water. The sample will then be added a few drops of 3 N hydrochloric acid that will
be sufficient to dissolve the bone sample. The sample will be added with 100 mL of
water, 15 mL of 1 N sodium hydroxide and 300 mg of hydroxyl naphthol blue. The
prepared solution will be titrated with 0.05 M edetate disodium VS (10g/500 mL water)
until the solution is a distinct blue in color. Each mL of 0.05 M edetate disodium is
equivalent to 5.004 mg of CaCO3 (USP-NF 2007).
The application of the sodium hydroxide is wide and found in different areas of
usage. The most widely and general application is its use as a chemical or in
manufacturing of other chemicals. It is also used as a strong base. The solution of sodium
hydroxide is very cheap and easy to carry and thus when needed in bulk it is used as
aqueous solution. The neutralization of the acidic material and the petroleum products is
also done through sodium hydroxide (SodiumHyroxide 2006).
Endrate (Edetate Disodium Injection, USP) is a sterile, nonpyrogenic,
concentrated solution of edetate disodium in water for injection which as a result of a pH
adjustment with sodium hydroxide contains varying amounts of disodium and trisodium
salts. After dilution, it is administered by intravenous infusion.
Each mL contains edetate disodium, anhydrous 150 mg. May contain sodium
hydroxide for pH adjustment, pH is 7.0 (6.5 to 7.5). Edetate disodium is classified as a
clinical chelating agent for emergency lowering of serum calcium in hypercalcemia(NLM
2006).
A calgamite indicator, often referred to as cal-red is used because it produces a
wine red color when bound to a metal ion. Upon further deprotonation, the indicator will
change from red to blue (pK2 = 8.1) forming HIn2-and then from blue to orange

(pK3=12.4) forming In3-. The calgamite indicator will form a wine-red complex with
metal ions. This is illustrated in the following reaction:
Ca2+ + In3- Ca-In(orange)

(wine red)

To quantiatively determine the amount of calcium present, a standardized solution

of EDTA is used as a titrant. The EDTA binds calcium much more strongly (pKf =10.69)
than does calgamite, allowing the following reaction to occur:
Ca-In- + EDTA Ca-EDTA2- + HIn2(wine red)

(blue)

Titration of the Ca-In- solution to a blue color indicates that sufficient EDTA was
added to bind all of the Ca2+ and the titration has reached its end point (Dorey and
Draves 1998).
Acids are estimated quantitatively by methods analogous to those employed for
the quantitative estimation of bases, viz., by directly titrating an exact quantity of the
acid, acid salt, or other acidic substance with standard alkali solutions or by adding an
excess of the latter and determining the amount in excess by residual titration with
standard acidic solution. Direct titration is employed whenever practicable, since it
requires only one standard solution, the standard base and fewer measurements of
volume. Assaying acids the quantity of acid to be taken should be such that about 30 to
40 mL of the standard base will be consumed. A general principle that is recommended is
that the normality of the solution obtained by dissolving the acid sample be
approximately the same as that of the titrant (Knevel and Digangi 1977).
The liquid that should be titrated must be brought to room temperature before
titration as many indicators give different values at different temperatures unless directed
otherwise. Organic acids, phenolphthalein is frequently used as an indicator however
thymol blue, bromothymol blue and thymolphthalein are also employed (Knevel and
Digangi 1977).
3. Diluent
3.1. General Characteristic
Diluents also known as fillers are inert substances used to create the desired bulk,
flow properties, and compression characteristics in the preparation of tablets and

capsules. Diluents make the required bulk of the tablet when the drug dosage itself is
inadequate to produce tablets of adequate weight and size. Diluents are often added to
dosage formulations for secondary reasons like to provide better properties (Pharmpedia
2008). Common diluents are lactose, microcrystalline cellulose, starch, powdered sucrose
and calcium phosphate. Bulking agents are chosen by the cost to obtain the ingredients
and by the experience of the manufacturer with a certain kind of bulking agent (Ansel
and others 1999).
Diluents pass through certain tests which determine if they are capable of being
used as diluents. Common physical properties which are being screened are the following
particle size and size distribution, particle shape, bulk/tapped/true density, specific
surface area, crystallinity, moisture content and powder flow (USP 2007).
3.2. Powder flow
Powder flow is the ability of a powder to flow and is usually a one dimensional
characteristic of powders (Prescott and others 2000). Powder flow can be determined
through two general methods which are static angle of repose and dynamic angle of
repose. Static angle of repose is done by letting a powder flow from a funnel onto a
surface and measuring the angle of repose. Angle of repose is the angle formed between
the horizontal plane and a sloped line extending along the face of a heap formed by
pouring material onto the horizontal surface (Bodhmage 2006). Powder flow can be
characterized by the angle of repose which is produced after static angle of repose
length of height

method. Powder flow was calculated by the formula: tan (0.5) ( width of base)
angle of repose
interpretation
(degrees)
25 - 30
excellent powder flow
31 - 35
good powder flow
36 - 40
fair powder flow
41 - 45
passable powder flow
46 - 55
poor powder flow
56 - 65
very poor powder flow
66 - above
very very poor powder flow
Table 1. Interpretation of angles of repose

3.3. Bulk and Tapped density

The bulk density of a powder is the weight of the powder divided by the volume it
occupies, normally expressed as g/ml or kg/l (GEA 2010). Tapped density is the
maximum packing density of a powder or blend of powders achieved under the influence
of well-defined externally applied forces (Thomas 2010). Tapped density can be
measured by computing the volume of a compacted powder of a known mass. Results
from bulk density and tapped density can be interpreted through many formulas one
common formula which is computed through the formula: Hausner Ratio=
TAPPED
DENSITY
, then
BULKDENSITY

the value is compared with the corresponding values (USP-

NF 2007).
Hausner ratio
1 - 1.11
1.12 - 1.18
1.19 - 1.25
1.26 - 1.34
1.35 - 1.45
1.46 - 1.59
1.6 - above

interpretation
excellent
good
fair
passable
poor
very poor
very very poor

Table 2. Interpretation of Hausner ratios

3.4. Particle shape
Particle shape is the general shape of a particle in its smallest discrete unit which
is usually characterized as acicular, columnar, flake, plate, lath and equant. Spherical
shapes such as flake, plate and equant are the shapes which exhibit the highest quality
because they exhibit good flow and particle distribution properties. Irregular shapes such
as lath exhibit passable flow and particle distribution properties. Needle like shapes such
as columnar and acicular exhibit poor flow properties due to interlocking of particles and
have poor particle distribution (USP-NF 2007).
particle shape
flake, plate or equant
lath
columnar or acicular

interpretation
high quality
passable
poor

Table 3. Interpretation of Particle Shape

3.5. Particle size
Particle size is the minimum size a certain coarse or granular particle can exist
without being chemically affected (Brittain 2002). Particle size can be determined by
microscopy and by estimation through analytical sieving. Analytical sieving is done by
stacking different sized sieves where the sieves with the largest apertures are on top while
the sieves with the smallest apertures are at the bottom. Particles must have a two
dimensional profile smaller than the sieve aperture in order to pass through a particular
sieve, a mean sieved diameter is calculated. The weight of particles on each sieve is
determined the mean sieved diameter represents a mass distribution (Martin 1993).
Powders are characterized into sub categories based on their particle sizes (Ansel and
others 1999).

interpretation
very coarse
Coarse

Sieve number
8
20
all
particles
pass
all particles
pass

moderately
coarse

40

60
not more
than 20 %

80

100

not more
than 40 %
all
particles
pass

Fine

not more than

40 %
all particles
pass

very fine

not more
than 40 %
all particles
pass

Table 4. Interpretation of Particle Size

3.6. Moisture content

Moisture content is the amount of water absorbed within a powder and can be
determined by loss on drying which is a procedure that determines the amount of volatile
matter of any kind that is driven off under the conditions specified. The ideal moisture

retention of calcium carbonate is that it must not lose 2% of its weight after it has been
dried at 200oC for four hours (USP-NF 2007).
weight loss
interpretation
below 2%
ideal moisture retention
2% and above
poor moisture retention
Table 5. Interpretation of Moisture content

4. Related Studies
A study by Phirapinyo done in 2006 and others focused on the calcium content in
the bones of giant seaperch (Lates calcarifer Bloch) and hoki (Macruronus
novaezelandiae) determined through calcium assay derived from USP 24. The results of
the study showed that the total calcium content in all fish bones varied from 28-32%
which is similar to another study referred to in this study which has results of 37.138.6%.
A study conducted by the USP Pharmacopeia done in 2007 to determine general
types of tablet or capsule diluents and the results stated different general types of
substances that are capable of either a capsule or tablet diluents such as Calcium
Carbonate, Calcium Phosphate, Dibasic Calcium Phosphate, Tribasic Calcium Sulfate
and more. The study used the following physical properties to screen a substances
capability as a general diluent: particle size and size distribution, particle shape,
bulk/tapped/true density, specific surface area, particle shape, particle size, moisture
content, powder flow, solubility and compaction properties for tablet dosage forms.
A study conducted by Taylor and others in 2000 used different methods to test
powder flow. The methods which were used were vibrating spatula, angle of repose,
percent compressibility index, critical orifice diameter, avalanching and principal
component analysis.
A study conducted by Bodhmage in 2006 to determine the correlation between
physical properties and flowability indicators for six fine powders specifically
NutraSweet Brand sweetener (Aspartame), Respitose ML001, Alpha-D-Lactose
monohydrate, Methocel (R) F50 Premium Hydroxypropyl Methylcellulose (HPMC), predried placebo pharmaceutical granulate and common pastry flour. The study used angle
of repose method, avalanching method and Electrical Capacitance Tomography to

determine flow properties. The angle of repose method consisted of placing a glass funnel
of opening 0.9 cm at a height of 6 cm from the surface, the powder was then allowed to
pass through and the angle of repose was determined both manually and through digital
imaging. Tapped density was determined by using a single platform density tester at a
setting of 300 taps per minute and the volume of the powder was checked every ten taps
until the volume had no significant change. True density was measured using helium gas
pyncnometry. Moisture content was determined using a halogen moisture analyzer where
the powders are continuously weighed while it is heated to remove the moisture. Particle
shape was determined by stereo microscopy at 90X magnification and scanning electron
microscopy at 200X and 500X magnifications.

Chapter 3
METHODOLOGY
I. List of Materials and Equipment
1. Chemicals:
Hydroxy Napthol Blue
Edetate Disodium
Hydrochloric Acid

Water
Sodium Hydroxide

2. Materials & Equipment:

Desiccator
Oven
Analytical Balance
Furnace
Table Top balance
No. 40, 60, 80, 100 mesh sieve
Mortar and Pestle
Digital Camera
Burette
500, 250, 50 mL Beaker
Glass Funnel
Light Microscope
10 mL Graduated Cylinder weighing paper
Milkfish and mangrove jack bone sample
II. Methods:
1. Procurement of Chemicals and Materials
Hydroxy napthol blue and edetate disodium were ordered and purchased from the
Chemistry Department of University of San Agustin. All other chemicals will be
requested from the chemistry stockroom of PSHS-WV. Laboratory equipment and
facilities will be requested from Philippine Science High School Western Visayas.
2. Collection of Milkfish and Mangrove Jack Samples
Fishes were procured from the fishpond of J.J.L. Corporation. An experienced
fisherman collected multiple milkfish and mangrove jack (Lutjanus argentimaculatus)
samples. 20 milkfish samples and 15 mangrove jack samples were de-boned.
3. Preparation of Milkfish and Mangrove Jack Bone Samples
3.1 Drying of Milkfish and Mangrove Jack bones
The fish bones were air dried for 168 hours. The 10 g of fish bones were
placed in a crucible and were heated in a furnace at 450oC until it became white
ash. The milkfish and mangrove jack bone samples were removed from the
furnace and were properly kept in a dessicator.
3.2 Dry Granulation and Segregation of Milkfish and Mangrove Jack Bones

The bone samples were crushed in a granulator until it was passed through
a sieve 40. The 4 g of powder bone sample was used in the tests for bulk density,
tapped density and powder flow was the same for the three tests and was carried
over to particle size. A 1 g separate amount of powder sample was segregated for
the calcium assay, moisture content test and particle shape.
4. Assay of Total Calcium Carbonate of Fish Bone Samples
The method was derived from USP manual 2007 edition. Bone sample of
200 mg which was previously dried was transferred to a 250 mL beaker. The
sample was moistened thoroughly with 3 mL water. The bone sample was
dissolved after adding 2 mL of 3 N hydrochloric acid. The sample was added with
100 mL of water, 15 mL of 1 N sodium hydroxide and 300 mg of hydroxyl
naphthol blue. The prepared solution was titrated with 0.05 M edetate disodium
VS which was previously prepared after adding 10g of EDTA to 500 mL water.
The solution was titrated until it is distinct blue in color. Each mL of 0.05 M
edetate disodium was equivalent to 5.004 mg of CaCO3.
5. Analysis of Powder Bone Sample Physical Properties
5.1 Bulk Density
The powder bone sample was passed through a No. 18 screen. 500 mg of
powder was passed through conical wax paper and then dropped into a dry 10 mL
cylinder without being compacted. The apparent unsettled volume was recorded.
The bulk density was calculated in grams per mL.
5.2 Tapped Density
The powder fish bones were through a No. 18 screen. 500 mg of powder
was passed through conical wax paper and then dropped into a dry 10 mL
cylinder. The cylinder was tapped and the volume was checked after three taps.
The tapped density was calculated in grams per mL.
5.3 Particle Shape
The 50 mg powder fish bones were placed on a clean glass slide. The slide
was placed under a light microscope. The particle shape was described based on
approximate shape
5.4 Powder Flow
A glass funnel six centimeters from the work surface was poured with 20
mL of bone sample. The height and the base of the conical heap were measured.

The angle was then computed by using the equation. The angle of repose was then
characterized based on the angle.
5.5 Moisture Content
A shallow weighing glass was dried for 30 minutes. A weighing paper was
used to weigh 100 mg of powder bone sample. The samples were placed inside
the oven at 200oC for four hours. The sample was allowed to reach room
temperature in a desiccator before being weighed again. The sample was then
weighed. The sample must have not lost more than 2.0% of its weight.
5.6 Particle Size
The 18.2 g of the powder bone sample was passed through four different
sieves No. 40, No. 60, No. 80, No. 100 placed on top of each other respectively.
The weight and percentage of powder on each sieve and in the collection pan was
calculated. Then the cumulative weight percentage of the powder that is finer than
the aperture was calculated

6. Disposal of Chemicals and Biological Waste

6.1 Biological Waste
All unused parts of the milkfish specifically the innards and some of the
organs were properly disposed in the biological waste bin. None of the biological
waste that arose from this research is deemed bio-hazardous.
6.2 Chemicals
All chemicals used in the research were not hazardous and were casually
disposed in the chemical waste bin according to MSDS standards.

Chapter 4
Results and Discussion
This study aimed to determine the physical properties and calcium carbonate content of
milkfish and mangrove jack bones as a pharmaceutical diluent. This study tested only the
following parameters: calcium carbonate content, particle size, particle shape, powder flow,
moisture content, bulk density and tapped density.
A) Results
Calcium

Particle

Particle

Powder

Moisture

Bulk

Tapped

Carbonate

Size
Sieve

Shape

Flow

Content

Density

Density

40:

0%
Sieve

60: flake and

0.37

0.65

g/mL

g/mL

Mangrove
Jack
45.12%

22.76%
plate
Sieve 80:

33.82o

2.75%

14.92%
Sieve 100:
8.33%
Sieve 40:
Milkfish

0%
Sieve
41.78%

60: flake and

19.43%
plate
Sieve 80:

42.88o

5.28%

0.35

0.54

g/mL

g/mL

18.18%
Sieve 100:
9.58%
Table 6. Calcium carbonate content and physical properties of milkfish and mangrove jack bones
as diluent

Mangrove jack bone sample contains more calcium carbonate compared to the
milkfish bone sample.
Mangrove jack and milkfish particle size is in between fine and very fine.
Milkfish and mangrove jack bone sample both exhibited a majority of flake and
plate shaped particles (Appendix B).
Mangrove jack bone sample exhibited an angle of repose characterized as good.
Milkfish exhibited an angle of repose characterized as passable. Mangrove jack has better
powder flow properties than milkfish.
Milkfish bone sample contains more moisture compared to mangrove jack bone
sample. Mangrove jack is better at not accumulating moisture than milkfish.
The bulk density and the tapped density of mangrove jack bone sample is very,
very poor quality. The bulk density and the tapped density of milkfish bone sample are of
poor quality.
B) Discussion
After the ashing of both milkfish and mangrove jack bones, we have observed that
ashed mangrove jack bones have a relatively darker color than that of the ashed milkfish
bones. It has only been observed that ashed mangrove jack bones flow much better than
ashed milkfish bones when it is poured for storage into different containers.

The calcium content in milkfish 41.78% and in mangrove jack is 45.12%. The
results did not reach the percentage of 90% calcium carbonate which is the minimum
calcium carbonate percentage content in common co processed diluents. The calcium
content found in both milkfish and mangrove jack is greater than the calcium content
found in two other fishes in the study of Phiraphinyo and others conducted in 2006. The
two fish which they used were the hoki and the giant seaperch with calcium carbonate
content percentages of 31.8% and 28.1% respectively. The implications of these results
are that most fish bones are not purely made of calcium carbonate and may contain other
substances which may decrease the quality of a diluent. These results are close to the
results of a related study but are even more desirable because of higher calcium carbonate
content in milkfish and mangrove jack.
The particle size of milkfish and mangrove jack are relatively larger compared to
most of the powders used in a study by Bodhmage in 2006. Bodhmage used the following
powders: aspartame which is very fine, ML001 which is also very fine, Lactose which is
also very fine, HPMC is also very fine, flour is also very fine and placebo granulate is
fine. The implications of these results are that milkfish and mangrove jack are larger in
Bodhmage.
The particle shape, plate and flake exhibited by both milkfish and mangrove jack
are the highest quality shapes because they contribute to good powder flow properties.
The particle shapes in a study by Bodhmage in 2006 are lower quality than those of
milkfish and mangrove jack. The following powders were analyzed in the study of
Bodhmage: flour is irregular, HPMC was spherical and elongated, ML001 were spherical
and irregular, lactose was irregular, placebo granulates was spherical and aspartame was
needle like. The implications of these results are that fish bones or other animal bones
may have a structure which is good for use as a diluent.
Mangrove jack has a powder flow angle of repose of 33.82 o characterized as
good. Milkfish has a powder flow angle of repose of 42.88 o characterized as passable.
Having a lesser angle of repose means that the when formed into a tablet the particles will
have a lesser tendency to separate when formed into certain shapes. The angle of repose
of mangrove jack is in the range of 31 o-35o. The angle of repose of milkfish is in the
range of 41o-45o. Mangrove jack is two intervals below the highest quality range while
milkfish is 4 intervals below. A study conducted by Taylor and others in 2000 to

determine the angle of repose of certain substances. Their results showed that pure
calcium carbonate has an angle of repose of 32 o. Mangrove jack bones has the closer
angle of repose to pure calcium carbonate with only a difference of 1.82 o, while milkfish
has an angle of repose with a difference of 10.88 o compared to pure calcium carbonate.
The results of this parameter are quite unexpected because milkfish and mangrove jack
have good particle shape which should contribute to excellent not passable powder flow.
Moisture content found in milkfish is 5.28% and in mangrove jack is 2.75% were
in excess compared to the standard 2%. The two fish samples hold an excess amount of
moisture compared to standard and may cause the particles to not form well. A study
conducted by Phiraphinyo and others in 2006 had result of moisture content in two fishes
specifically the hoki which was 12. 4% and giant seaperch was found to be 11.3%.
Moisture content in milkfish and mangrove jack is relatively closer to the standard 2%
target than hoki and giant seaperch. None of the fishes though have reached the standard.
These results imply that if turned into a diluent the fish bone ingredients will collect
unnecessary moisture and may contribute to the spoiling or disintegration of the product.
The excess moisture in the fish bone samples may have been caused by the more than
roughly 50% of unknown substance in the fish samples.
Bulk density and tapped density is very, very poor in mangrove jack while in
milkfish is very poor based on Hausner Ratio equation. The bulk density and tapped
density of milkfish and mangrove jack interpreted as Hausner Ratios, compared to the
powders used in the study by Bodhmage conducted in 2006 the values are relatively
close. The Hausner ratios of powders used by Bodhmage are the following: aspartame
with a failed value, HPMC with poor value, placebo granulates with fair value, lactose
with poor value, ML001 with very, very poor value and flour with a poor value. These
results have contributed that powder flow is not of the highest quality. Bulk density and
tapped density characterized as Hausner ratio is a very important determinant of powder
flow.

Chapter 5
Conclusion and Recommendation
This study aimed to:
1.) Measure the calcium carbonate content of milkfishs and mangrove jacks bones.
2.) Measure the physical properties of milkfishs and mangrove jacks bones to be used as
diluents, in terms of:
a) particle size
b) particle shape
c) powder flow
d) moisture content
e) bulk density
f) tapped density
3.) Compare the physical properties of milkfishs and mangrove jacks bones as diluents
with the pharmaceutical standard.
A) Summary of Results
1.)

The calcium carbonate content of the mangrove jack bone samples and milkfish

bone samples are 45.12% and 41.78% respectively.

2.)

Mangrove jack bone samples have particle size in between fine and very fine,

particle shapes of flakes and plates, powder flow angle of repose of 33.82o, moisture content of
2.75%, and bulk and tapped densities very, very poor quality.

Milkfish bone samples have a particle size in between fine and very fine, particle
shapes of flakes and plates, powder flow angle of repose of 42.88 o, moisture content of 5.28%,
and bulk and tapped densities very poor quality.
B) Conclusion
Based on the results of our tests, both Mangrove Jack and Milkfish bones do not pass the
standards set by USP for pharmaceutical tablet diluents.

C) Recommendation
This study can be improved if an automatic tapped density tester is used for more
accurate measuring of the tapped density of the fish bones. The study can be improved if more
accurate equipment will be used to determine exact calcium carbonate and other components in
the fish bones. This study can be improved if microscope and microscope camera with a higher
resolution and magnification will be used. This study can be improved by using an automatic
sieve tapper. This study can be improved if a more precise instrument to measure angle of repose
is used.
Based on our results, future studies can use other kinds of fish and tested if the bones of
other fishes can pass the pharmaceutical standard for tablet diluents set by the USP
Future studies can study further if the bones of milkfish and mangrove jack can be used
as a cheaper source of calcium to be made as calcium supplements

Literature Cited
Amidon G., Peck G., Block L., Moreton R., Katdare A., Lafaver R. 2007. Proposed New USP
General Information Chapter Excipient Performance[Internet]. [cited 2009 September
21]. Available from http://www.usp.org/pdf/EN/USPNF/Vol33No7Stimuli.pdf
Ansel H., Allen L. Jr., Popovich N., 1999. Pharmaceutical Dosage Forms and Drug Delivery
Systems 7th Edition. Lippincott Williams & Wilkins. 351 West Camden Street Baltimore,
Maryland 21201-2436 USA.
Bagarianao T., October 1999. Ecology and Farming of Milkfish. Tigbuan, Iloilo, Philippines.
Bodhmage A. July 2006. Corelation between physical properties and flowability indicators for
fine

powders

Available:

http://library2.usask.ca/theses/available/etd-

07032006115722/unrestricted/Abhay_thesis.pdf via the INTERNET. Accessed July 2010

Brittain H. 2002. Particle-Size Distribution, Part III: Determination by Analytical Sieving
available:
http://pharmtech.findpharma.com/pharmtech/data/articlestandard//pharmtech/502002/409
75/article.pdf via the INTERNET. Accessed July 2010
Calcium Carbonate Organization. 2008. Calcium Carbonate [Internet]. [cited 2010 January 24].
Available from http://www.ima-eu.org/fileadmin/cca/CalCarb05.pdf
Dorey., Draves., 1998.[Determination of Calcium ions by EDTA Titration. Internet]. [cited 2010
June 10]. Available from
http://chemistry.uca.edu/Projects/Quant98/calcium-edta-titrn 98.pdf via the INTERNET
FishBase Organization.2009. Lutjanus Argentimaculatus[Internet]. [cited 2009 November 5].
Available
from
http://fishbase.org/Summary/speciesSummary.php?
ID=1407&genusname=Lutjanus&speciesname=argentimaculatus&lang=English
GEA Niro. 2010. Bulk Density Considerations Available:
http://www.niro.com/niro/cmsdoc.nsf/WebDoc/ndkw6dknxs via the INTERNET.
accessed July 2010
Isimi C., Nasipuri R., Ojile J., Ibrahim Y., Emeje M. 2003. Effects of the diluents type

on compressional characteristics of the mixed stem bark extract of Anegeissus leiocarpus

and Prosopis africana tablet formulation[Internet]. [cited 2009 November 5]. Available
from http://www.sssbjt.hr/acphee/Isimi.pdf via the INTERNET accessed: July 2009
Linda Houtkooper, Veronica A. Mullins. 2002. Calcium Supplement Guidlines[Internet].
[cited 2009 December 3]. Available from http://ag.arizona.edu/pubs/health/az1042.pdf
Knevel A., Digangi F., 1977. Jenkins Quantitative Pharmaceutical Chemistry 7th Edition.
McGraw-Hill, Inc. Merriam Webster Bookstore, Inc. 864-870 N. Reyes Sr. Ave.,
Sampaloc, Mla.
Martin A., Bustamante P., Chun A. 1993. Physical Pharmacy 4th Edition. Lippincott Williams &
Wilkins, 351 West Camden Street, Baltimore, Maryland, 21201-2436 USA. C & E
Publishing Inc.
Mullins V, Houtkooper L. 2004. Calcium Supplement Guidelines. The University of Arizona
College of Agriculture Tucson, Arizona [Internet].[cited 2008 Aug 25] 85721. Available
from: http://ag.arizona.edu/pubs/health/az1042.pdf
(NIH) National Institute of Health. 2009: Calcium Fact Sheet: Available:
http://ods.od.nih.gov/factsheets/calcium.asp: via the INTERNET:
Bethesda, Maryland. Accessed: July 13, 2009.
(NSDB) National Science Development Board. 1978: Milkfish (Bangos) as Food: Bicutan,
Taguig, Metropolitan Manila, Philippines
Dr. Nolting M. 2006: Rapid Study of the European market for Philippine Milkfish: The Trade
Policy and Trade Promotion Project. 10th Floor, German Developmant Center, PDCDP
Bank Center Building, V.A. Rufino cor. L.P. Leviste Streets, Salcedo Village, Makati
City, Philippines.
Ong J. 2008: Laboratory Manual Manufucturing Pharmacy: University of San Agustin
College of Pharmacy and Medical Technology. Iloilo City, Phillipines.
(PUA) Pharos University in Alexandria. 2009. Tablet coating[Internet]. [cited 2010 February
13].
Available
from
http://www.pua.edu.eg/PUASite/uploads/file/Pharmacy/fall2009/PHR312/week5/Micros
oft%20Word%20-%20Lect%205%20DDF%20II%2009-10.pdf
(PUA) Pharos University in Alexandria. 2009. Tablet excipients[Internet]. [cited 2010 February
13].
Available
from
http://www.pua.edu.eg/PUASite/uploads/file/Pharmacy/fall2009/PHR312/week3/Micros
oft%20Word%20-%20lec%203%2009-10%20pdf.pdf
(PUA) Pharos University in Alexandria. 2009. Tablet manufacturing[Internet]. [cited 2010
February 13].
Available
from
http://www.pua.edu.eg/PUASite/uploads/file/Pharmacy/fall2009/PHR312/week2/Micros
oft%20Word%20-%20DDF%20II%20Lecture%202%2009-10.pdf

(PUA) Pharos University in Alexandria. 2009. Types of tablets[Internet]. cited 2010

February 13].
Available

from

http://www.pua.edu.eg/PUASite/uploads/file/Pharmacy/fall2009/PHR312/week6/Micros
oft_Word_-_Lecture_6_09-10.pdf
Phiraphinyo P., Taepakpurenat, S., Lakkanatinaporn, P., Suntornsuk, W., Suntornsuk, L.
March- April 2006. Physical and Chemical Properties of Fish and Chicken
Bones as Calcium Source for Mineral Supplements: Vol. 28 No.2. 328-335: Available:
http://sjst.psu.ac.th/journal/28_2_pdf/11_calcium_source.pdf via the INTERNET:
Accessed 2009 July 1.
Prescott J., Barnum R. 2000. On Powder Flowability. Available:
http://www.jenike.com/TechPapers/on-powder-flowability.pdf via the INTERNET:
Accessed 2010 July 10
(PHB) Prevention Health Books. 2000: Vitamin Cures The Ultimate Compendium of Vitamin
and Mineral Cures with More than 500 remedies for Whatever Ails You!: St. Martins
Press. St. Martins Paperbacks, 175 Fifth Avenue, New York, NY 10010,
U.S.A.1998,2000
Saigal N., Baboota S., Ahuja A., Ali J. 2009. Microcrystalline Cellulose as a Versatile Excipient
in Drug Research[Internet]. [cited 2010 January 25]. Available from
http://www.jyoungpharm.in/article.asp?issn=09751483;year=2009;volume=1;issue=1;spage=6;epage=12;aulast=Saigal
Siegelbaum J. 2009 : Minerals: Available:
http://gicare.com/For-physicians/minerals.aspx. 423 N, 21st Street. 2008. Camp Hill,
PA 17011 via the INTERNET: Accessed July 13,2009
(SEAFDEC) Southeast Asian Fisheries Development Center. July 2009. Training Handbook on
Rural Aquaculture. Tigbuan, Iloilo, Philippines. 296 p.
Sodium Hydroxide Organization. 2009. Sodium Hydroxide[Internet]. [cited 2010 April 27].
Available from http://www.sodiumhydroxide.org
Taylor M., Ginsburg J., Hickey A., Gheyas F. 2000. Composite Method to Quantify Powder
Flow as a Screening Method in Early Tablet or Capsule Formulation Development.
Available from http://www.aapspharmscitech.org/articles/pt0103/pt010318/pt010318.pdf
via the INTERNET: Accessed July 10 2010
U.S. Environmental Protection Agency. 2002. Hydrochloric Acid (Hydrogen
Chloride)[Internet].
[cited
2010
January
24].
Available

from

http://www.epa.gov/ttn/atw/hlthef/hydrochl.html
U.S. National Library of Medicine.2008. Edentrate(edentate disodium,anhydrous) Injection,
Solution. [Internet]. [cited 2010 April 27].
Available from http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=3269
(WBI) World Book Incorporated. 2000. World Book Millenium 2000. Composition of Bone. 233
North Michigan Chicago, IL, 60601.
Vanzi S. 2005: Nothing is Wasted in Bangus (Milkfish) Now:

Available: http://newsflash.org/2004/02/si/si001806.htm.via the INTERNET. Accessed

July 1, 2009

Appendix A
Raw Data

Mangrove
Jack
Trial
1
2
3
mean

EDTA (mL)
18.3
17.6
18.2

CaCO3 content (mg)

91.5732
88.0704
91.0728

CaCO3 percent (%)

45.7866
44.0352
45.5364
45.1194

Trial
EDTA (mL) CaCO3 content (mg)
1
17.2
86.0688
2
16.6
83.0664
3
16.4
82.0656
mean
Raw Data for Calcium carbonate Content

CaCO3 percent (%)

43.0344
41.5332
41.0328
41.8668

Milkfish

Mangrove jack
mass of the powder left on
the sieve (g)
Total mass
Trial placed (g)
1
17.93
2
17.93
3
17.69
mean 17.85
%

sieve 40
0.00
0.00
0.00
0.00
0.00

sieve
60
4.34
3.71
4.14
4.06
22.76

sieve
80
2.45
3.04
2.50
2.66
14.92

sieve
100
1.72
1.44
1.30
1.49
8.33

sieve
60
3.71
3.66
3.36
3.58
19.43

sieve
80
3.31
3.28
3.45
3.35
18.18

sieve
100
1.59
1.72
1.98
1.76
9.58

Milkfish
mass of the powder left on
the sieve (g)
Trial
1
2
3
mean
%

Total mass
placed (g)
18.49
18.42
18.32
18.41

Raw data for particle size

sieve 40
0.00
0.00
0.00
0.00
0.00

Mangrove Jack
Trial
1
2
3
mean

Base (cm)
6.6
6.8
6.9

Height (cm)
2.2
2.3
2.3

Angle of repose (o)

33.69
34.08
33.69
33.82

Base (cm)
6.3
6.8
6.5

Height (cm)
2.9
3.1
3.1

Angle of repose (o)

42.63
42.36
43.65
42.88

Milkfish
Trial
1
2
3
mean
Raw data for Powder Flow

Mangrove jack
Trial
1
2
3
mean

Initial mass (g)

0.1007
0.1008
0.1006

final mass (g)

0.0982
0.0982
0.0974

percent loss (%)

2.48
2.58
3.18
2.75

Trial
Initial mass (g)
1
0.1007
2
0.1002
3
0.1003
mean
Raw data for Moisture Content

final mass (g)

0.0959
0.0950
0.0944

percent loss (%)

4.77
5.19
5.88
5.28

Milkfish

Mangrove Jack
Trial
1
2
3
mean

mass (g)
0.50
0.50
0.50

volume (mL)
1.40
1.30
1.30

density (g/mL)
0.36
0.38
0.38
0.37

Trial
1
2
3
mean

mass (g)
0.50
0.50
0.50

volume (mL)
1.50
1.50
1.30

density (g/mL)
0.33
0.33
0.38
0.35

Milkfish

Raw data for Bulk Density

Mangrove Jack
Trial
1
2
3
mean

mass (g)
0.50
0.50
0.50

volume (mL)
0.80
0.70
0.80

density (g/mL)
0.63
0.71
0.63
0.65

Trial
1
2
3
Mean

mass (g)
0.50
0.50
0.50

volume (mL)
1.00
0.90
0.90

density (g/mL)
0.50
0.56
0.56
0.54

Milkfish

Raw Data for Tapped Density

Appendix B
Plates

Plate 1: Mangrove Jack particle shape

Plate 2: Mangrove Jack particle shape

Plate 3: Mangrove jack particle shape

Plate 4: Milkfish particle shape

Plate 5: Milkfish particle shape

Plate 6: Milkfish particle shape

Plate 7: Sieving of Milkfish Sample

Plate 8: Calcium carbonate assay of mangrove jack sample

Plate 9: Weighing of milkfish sample for moisture content

Plate 10: Preparation of powder flow funnel setup

Plate 11: Measuring of volume for tapped density of mangrove jack sample