Use of rice husk as natural adsorbent to treat wastewater containing iron II sulfate and its toxicity test on nile tilapia

THAI NGUYEN UNIVERSITY
UNIVERSITY OF AGRICULTURE AND FORESTRY

DEASY AMALIA SARI

BACHELOR THESIS
USE OF RICE HUSK AS NATURAL ADSORBENT TO TREAT WASTEWATER
CONTAINING IRON II SULFATE AND ITS TOXICITY TEST ON NILE TILAPIA

Study Mode

: Full-Time

Major

: Environmental Science and Management

Faculty

: Advanced Education Program

Batch

: 2014-2018

Thai Nguyen, September 2018


DOCUMENTATION PAGE WITH ABSTRACT
Thai Nguyen University of Agriculture and Forestry
Degree Program

Bachelor of Environmental Science and Management

Student Name

Deasy Amalia Sari

Student ID

DTN1454290076

Thesis Title

Use of Rice Husk as Natural Adsorbent to Treat Wastewater
Containing Iron II Sulfate and Its Toxicity Test on Nile Tilapia

Supervisor (s)

Dr.-phil. Dipl.-Ing.agr. Arinafril
Prof. Tran Van Dien

Abstract :
The problem of clean water now becoming a global concern. Water is a source of life and
is used in various ways such as for cooking, bathing, washing, farming, and other
activities. The needs of clean water become difficult to be fulfilled due to the presence of
water problems. One of the water problems occur is water pollution where the water
contains harmful substances such as heavy metals and the number is exceeds the maximum
contaminant level of iron that allowed in the water. One of heavy metals that often found
in the water is iron (Fe). Maximum allowed iron content in the water is by not exceeding
0.1mg/L. High iron content in the water makes turbid water has smells, causes rust on

household appliances, and it affects to the health problems. Histopathology examination on
fish gill indicates that excessive levels of Fe can damage the tissues of the fish gills. The
alterations detected were as Telangiectasia, Fusion of Secondary Lamellae, Epithelial
Proliferation of Secondary Lamellae and Congestion. Treatment to the water is must be
carried out in order to decrease the excess iron content. However, the processing is quite
difficult to do and requires a large cost. The use of rice husk is a good alternative way to

i


treat the water containing high iron content. Rice husk as a natural adsorbent has the ability
to absorb heavy metals. Adsorbent test treatment resulted in the decrease of iron levels that
contain in the water. It showed that rice husk as a natural adsorbent has the ability to
absorb Fe ions and effective in handling water problems including water that contains high
of iron content.
Rice husk, bioadsorbent, water pollution, histopathology,
Keywords
Oreochromis niloticus.
Number of Pages

46

Date of Submission
1.
Supervisor’s
Signature

2.

ii

ACKNOWLEDGEMENT
From bottom of my heart, I would like to express my deepest and solid
gratitude to ALLAH SWT, Almighty God for giving me the strength and courage as
the time gone by, for fading away doubts and leading me to finish this bachelor thesis.
I would like to express my sincere gratitude and deep regards to my supervisor:
Dr.-phil. Arinafril of Sriwijaya University, Indralaya, Indonesia, who guided me
wholeheartedly when I conducted this research, gave additional knowledge, worthy
indications, comments and guidance from the start to the end of this study.
I also want to thank my second supervisor, Prof. Tran Van Dien, for his
supervision, encouragement, advice, and guidance in writing this thesis.
Likewise, I would like to thank Ph.D. Dr. Krisna Murti in Department of
Anatomical Pathology, who was very patiently assisted me with the histopathology
examination for this study.
Furthermore, an acknowledgement also goes to the Rector of Sriwijaya
University, Prof. Dr. Ir. H. Anis Saggaf, MSCE., for acknowledging the internship
acceptance.
I would also like to say thank to the Dean of Faculty of Agriculture in Sriwijaya
University, Prof. Dr. Ir. Andy Mulyana, M. Sc., who gave me the permission to use
all the necessary facilities for the experiment that has been conducted at Aquaculture
Laboratory and Fish Product Technology Laboratory, Faculty of Agriculture,
Sriwijaya University, Inderalaya Campus.
In addition, to Prof. Dr. Mohammad Amin, S.Pi., M.Si., Prof. Mochamad
Syaifudin, S.Pi., M.Si., PhD, and Prof. Dade Jubaedah, S.Pi., M.Si., from Budidaya
Perairan, Faculty of Aquaculture, Sriwijaya University, Inderalaya Campus. Thank
you for the additional knowledge and guidance during the experimental time.

iii

I also place my gratitude to Mrs Nurhayani, Mrs Ana, Ms Naomi, other staffs
and friends in Aquaculture Laboratory and Fish Product Technology Laboratory,
Sriwijaya University. Thank you for helping, providing me necessary equipment as
well as teaching additional knowledge during my experiment at the laboratories.
The unconditional love from my family, to my parents, Hidayat and
Maydaria, my elder sister, Dewi Permatasari, my elder brother Muhammad
Alfarizi, and my younger sister, Metha Maya Sari, who gave me strength, support
and positive thoughts to finish what I have started and lead me to the successful
completion of this study.
Nevertheless, I would like to thank my best friends – Nur, Givanni, Rani,
Annisa, Ara, Dhiya and Ica; my bachelor thesis mates – Amana and Vidya; and all
Indonesian friends in the Vietnam. Thank you for the unending support and giving the
positive thoughts until I finish this study.

Thai Nguyen, 24th September, 2018

Student
Deasy Amalia Sari

iv


TABLE OF CONTENT
ACKNOWLEDGEMENT................................................................................................................. iii
LIST OF FIGURES ............................................................................................................................ 1
LIST OF TABLES .............................................................................................................................. 2
PART I. INTRODUCTION ................................................................................................................ 3
1.1.

Background and Rationale ................................................................................................... 3

1.2.

Objectives ........................................................................................................................... 4

1.3.

Research Questions and Hypotheses .................................................................................... 4

1.3.1. Research Questions ........................................................................................................... 4
1.3.2. Hypotheses ........................................................................................................................ 5
1.4.

Limitations .......................................................................................................................... 5

PART II. LITERATURE REVIEW..................................................................................................... 6
2.1.

Iron ..................................................................................................................................... 6

2.1.1. Iron (II) Sulfate ................................................................................................................ 7
2.2.

Rice Husk ............................................................................................................................ 8

2.3.

Natural Adsorbent ............................................................................................................... 9

2.3.1. Rice Husk as Adsorbent ................................................................................................. 10
2.4.

Test Species – Oreochromis niloticus ................................................................................. 10

2.5.

Histopathological Effects ................................................................................................... 11

PART III. METHODOLOGY ........................................................................................................... 13
3.1.

Place and Time .................................................................................................................. 13

3.2.

Equipment and Materials ................................................................................................... 13

3.3.

Fish preparation, Preparation of Fe2+ Solution and Adsorbent Preparation .......................... 14

3.3.1.

Fish Preparation ........................................................................................................ 14

3.3.2.

Preparation of Fe2+ Solution ...................................................................................... 14

3.4.

Methods ............................................................................................................................ 15

3.4.1.

Toxicity Experiment ................................................................................................... 15

3.4.2.

Heavy Metal Adsorption Test using Fish as Indicator ................................................. 15

3.4.3.

Adsorbent Adsorption Examination ............................................................................ 15

3.4.4.

Histopathology Examination ...................................................................................... 16

PART IV. RESULTS AND DISCUSSIONS ..................................................................................... 18
4.1. Preliminary Toxicity Test ....................................................................................................... 18
4.2. Heavy Metal Adsorption Test using Fish as Indicator ............................................................. 19
4.3. Adsorbent Adsorption Examination ........................................................................................ 22
4.4. Histopathological Observation of Fish Gills ............................................................................ 24

v


PART V. CONCLUSION ................................................................................................................. 36

REFERENCES ................................................................................................................................. 37
APPENDICES .................................................................................................................................. 42

vi


LIST OF FIGURES
Figure 1. Adsorbent Adsorption Result .................................................................23
Figure 2. Normal structure of gills ........................................................................24
Figure 3. Fish gill exposed to Iron (II)- sulfate (FeSO4) with concentration of
5.821mg/L without adsorbent treatment (x100) ......................................25
Figure 4. Fish gill exposed to 5.821mg/L iron (II)- sulfate (FeSO4) with
adsorbent treatment (x100) .....................................................................26
Figure 5. Fish gill exposed to 5.2389 mg/L iron (II)- sulfate (FeSO4) with
adsorbent treatment (x100) .....................................................................28
Figure 6. Fish gill exposed to 4.6568mg/L iron (II)- sulfate (FeSO4) with
adsorbent treatment (x100) .....................................................................29
Figure 7. Fish gill exposed to 6.4031mg/L iron (II)- sulfate (FeSO4) with
adsorbent treatment (x100) .....................................................................30
Figure 8. Fish gill exposed to to 6.9852mg/L iron (II)- sulfate (FeSO4) with
adsorbent treatment (x100) .....................................................................32


LIST OF TABLES
Table 1. Iron (II) Sulfate Property ................................................................................7
Table 2. Iron (II) Sulfate Potential Health Effects ........................................................8
Table 3. Rice Husk Property ........................................................................................9
Table 4. List of Equipment Used ................................................................................ 13
Table 5. List of Material Used .................................................................................... 14
Table 6. Preliminary Toxicity Test Result .................................................................. 18

Table 7. Fish Mortality............................................................................................... 20
Table 8. Effect of Iron (II) Sulfate towards Nile Tilapia in 24 hours ........................... 21
Table 9. Adsorbent Adsorption Examination Result ................................................... 23
Table 10. Gills Alterations ......................................................................................... 35

2


PART I. INTRODUCTION
1.1.

Background and Rationale
Heavy metals that lead to the water pollution is currently becoming a global

concern. Lack of clean water is often found in some places in the world. Needs of
clean water must be fulfilled and one of clean water sources is by using ground water
that obtained from well and borehole water. However, deficiency of using groundwater
commonly can cause several ailments to human health immediately while the
contaminants of gas as well as mineral exceeds the maximum allowed level (Suharno,
2017).
The presence of dissolved iron in the water can affect the stinking metal flavor,
cause the growth of iron bacteria, create reddish-brown staining on clothing, bathtub
and other equipment, or even blockage on pipelines. In addition, iron that has a
concentration greater than 25 mg/L will give a sense of metallic, astrinogent or
medicine taste. (Dharma, 2002). Maximum contaminant level of Iron that allowed in
the water is 0.3mg/L (U.S. Environmental Protection Agency, 2013). Some heavy
metals removal technologies such as chemical precipitation, reverse osmosis, ion
exchange, ultrafiltration, electrodialysis, as well as phytoremediation are generally
used for industrial wastewater management. Nevertheless, these heavy metals removal
methods are usually inexpensive and inadvisable (Ahalya, Ramachandra and

Kanamadi, 2003; Yus and Mashitah, 2014). Cost effectiveness is the main attraction of
metal biosorption, and it should be kept that way.
The use of bioadsorben as natural adsorbent material is one of the effective
ways to treat wastewater containing hazardous compound (Viera and Volesky, 2000).
One of natural bioadsorbents is Rice husk. Rice husk is a product in the rice milling
industry. It is also one of the most important agricultural residues in a big quantity
amount. On weight basis of the whole rice, it represents around 20% of the whole rice
produced (Daifullah, Girgis and Gad, 2003). In the study by Kumar and
Bandyopandhyay (2005), rice husk has been reported for their ability to bind metal
ions. The use of rice husk which is not utilized after the harvest time as a natural
adsorbent is a good alternative way to treat wastewater problems.
3


Biomass composed of cellulose, hemicellulose, protein, fat and lignin, can be
used as bioadsorbents. It composed of active groups in the form of phenolic, hydroxy,
carboxyl, amine, phosphate groups, which can be a simple treatmeant or as adsorbents
which have the ability to bind heavy metal ions. (Kim et al., 2005; Jhonson, Jain and
Prasad, 2008 cited in Lesmana et al., 2009). A huge amount of rice husk usually burnt
by in situ processs, produce CO2 and pollution in many forms. Hence, the use of rice
husk not individually would provide a low cost sorbent in activated carbon nor
synthetic in ion exchange and easily to find, but also maintain the environment from
numerous pollution problems (Kumar, 2009).
The aim of this study is to investigate the effectiveness of using rice husk as
natural adsorbent to treat wastewater containing iron (II) sulfate by conducting the
toxicity test on Nile Tilapia fish. To see how much the iron (II) sulfate that can be
adsorbed by the natural adsorbent through the adsorbent adsorption examination. To
determine the damage that might be occured by the pollutan (Fe), histopathological
examination will be conducted on fish gills organ.
1.2.

Objectives
This study was designed to assess the toxicological effects of iron (II)- sulfate

(FeSO4) on Oreochromis niloticus before and after the adsorbent was given as well as
to reveal the histopathological alterations on its gills.
1.3.

Research Questions and Hypotheses

1.3.1. Research Questions
The study was conducted to answer specific questions:
1. How does iron (II)- sulfate (FeSO4) affect the gills of Nile Tilapia before the
adsorbent is given?
2. How does iron (II)- sulfate (FeSO4) affect the gills of Nile Tilapia after the
adsorbent is given?
3. How is the effectiveness of rice husk for the removal of iron (II)- sulfate
(FeSO4)?

4


1.3.2. Hypotheses
Hypotheses 1:
H0 (Null Hypotheses): Exposure to iron (II)- sulfate (FeSO4) will not result in
change in gills histology of Nile Tilapia after the adsorbent is given.
HA (Alternative Hypotheses) : Exposure to iron (II)- sulfate (FeSO4) will result
in change in gills histology of Nile Tilapia after the adsorbent is given.

Hypotheses 2:

H0 (Null Hypotheses): Exposure to iron (II)- sulfate (FeSO4) will not result in
change in gills histology of Nile Tilapia before the adsorbent is given.
HA (Alternative Hypotheses) : Exposure to iron (II)- sulfate (FeSO4) will result
in change in gills histology of Nile Tilapia before the adsorbent is given.

Hypotheses 3:
H0 (Null Hypotheses): Rice husk is not effective for the removal of iron (II)sulfate (FeSO4).
HA (Alternative Hypotheses): Rice husk is effective for the removal of iron (II)sulfate (FeSO4).
1.4.

Limitations
There is a lack of experimental methodology of adsorbent testing directly to

the O. niloticus as well as lack of experimental results to the histopathological
effects on gills tissues of fish of iron (II)- sulfate (FeSO4) in the literature.

5


PART II. LITERATURE REVIEW
2.1.

Iron
Iron is the second most plentiful metal in the earthh's crust. Iron elements is

barely presence in nature, and usually in the form of ions Fe2+ and Fe3+ that
immediately combine with oxygen as well as sulfur containing compounds as an
accumulator of oxides, sulfides carbonate and hydroxides. Iron is often generally
found in nature, as its oxides forming (Elinder, 1986; Knepper, 1981).
The presence of iron in the environment is constant and as necessary

component to the animal as well as human. Applied for the synthesis of hemoglobin
and myoglobin in the muscle. Requirement of iron in the daily basis is provided from
food. When inadequacy disorders are happened, it can be treated with the use of iron
salts as a remedy. Iron can be found in almost all organs as well as tissues, in addition
the largest amount of iron are stocked in the liver and spleen. Iron salts captured
verbally do not poison, nevertheless can also deliver side effects such as obstipation
and vomiting (Beata, 2014).
An analysis of iron toxicity to the aquatic plants specifically on rice, stated
that the growth of aquatic reed species was found prevented by concentration of
1mg/L of total iron (Phippen et al., 2008). The function of iron toxicity within rice
involve excessive of Fe2+ from roots, acropetal translocation to the leaves, changing
color of rice leaves as well as yield loss (Becker and Asch, 2005). An excessively
level of iron infilrate into the human body move across the rate-limiting absorption
phase and develop into saturated. Those free irons get through physically into cells of
the brain, heart and liver. Whereas the disorder of oxidative phosphorylation from
free iron, ferrous iron is transformed to ferric irom that unbind hydrogen ions, hence
enlarging metabolic acidity. The free iron might also becomes lipid peroxidation that
results in particular loss to mitochondria, microsomes and other cellular organelles
(Albretsen, 2006).
Iron toxicity on cells has persuaded to iron mediated tissue injuries including
cellular oxidizing and decreasing mechanisms and its toxicity against intracelular
organelles like mitochondria and lyso-somes. An expansive of free radicals which are
able to cause capability cellular damage are delivered by plenty intake of iron.
6


Hydrogen free radicals that produced by iron are able to attack DNA, lead to cellular
damage, mutation as well as malignant transformations that will cause various
disease (Grazuleviciene et al., 2009).
Heavy metals are usually found in both environment and diet. While the

amounts are in allowed limits, they are necessary for maintaning good health. In
addition, if the amounts presence are above its allowed limits, they might become
dangerously toxic. Heavy metal toxicity will decrease energy levels as well as
damage the function of brain, lungs, liver, kidney, blood structure and other
important organs. If the exposure is in a long-term period of time, that might lead to
gradually physical progressing, muscular, as well as neurological degenerative
practices and affect several diseases such as multiple sclerosis, Parkinson's disease,
Alzheimer's disease and muscular dystrophy. Continuosly exposure of some metals
and its compounds might even lead to cancer (Jarup, 2003).
2.1.1. Iron (II) Sulfate
Iron (II) sulfate or Ferrous Sulfate is a greenish or yellow-brown crystalline
solid from synthetic origin and belongs to the pharmacological groups known as
hematological agents and iron salts. The main hazard is the threat to the environment
(“DrugBank,” n.d; “Cameo Chemicals,” n.d). Stabilized of iron (II) sulfate has a
natural composition such as ferrous sulfate stabilized with glycine and malic acid
(Salgueiro, Torti and Meseri, 2007). Iron (II) sulfate property as well as its potential
health effects displayed in Table 1 and Table 2.
Table 1. Iron (II) Sulfate Property
Chemical Name
Common Name
CAS NO.
Molecular Weight
Chemical Formula
Appearance
Solubility
Density
Boiling Point
Melting Point
Hazardous

Iron (II) sulfate (1:1), heptahydrate; sulfuric acid, iron (2+)
salt (1:1), heptahydrate.
Ferrous Sulfate
7720-78-7
278
FeSO4
Blue green crystals
48.6 g/100 g water @ 50C (122F)
1.90
> 300°C (> 572°F) Decomposes
57°C (135°F) Loses water
Yes

(Source: MSDS, 2000)
7


Table 2. Iron (II) Sulfate Potential Health Effects
Type of
Health Effect
Inhalation

Ingestion

Skin Contact

Eye Contact

Description

First Aid Measures

Irritation in the respiratory tract. Release to fresh air. Whenever
Symptoms might include coughing breathing is difficult, give an
and shortness of breath.
artificial respiration and oxygen.
Get medical attention for further
aid.
Low toxicity in small number but Generate vomiting immediately
higher dosages may cause nausea, as
directed
by
medical
vomiting, diarrhea, and black stool. personnel. Do not give anything
Pink urine discoloration indicates by mouth to an unconscious
strong iron poisoning. Lead to liver person. Get medical attention for
damage, coma, and death due to iron further medication.
poisoning.
Children
are
more
vulnerable to the iron toxic at smaller
concentration.
Skin irritation. Several symptoms such Rinse skin immediately with
as redness, itching, and pain.
plenty of soap and running water
for at least 15 minutes. Change
contaminated clothing and shoes.
Get medical attention. Wash
clothes and shoes thoroughly

before reuse.
Irritation, redness, and pain.
Flush eyes directly with plenty
of water for at least 15 minutes,
lifting lower and upper eyelids
occasionally and get medical
attention for further medication.

(Source: MSDS, 2000)
2.2.

Rice Husk
Rice husk is a product in the rice milling industry and it is one of the most

largely agricultural wastes in rice producing. It is approximately 600 million tons of
rice paddy is produced globally for each year. It represents on average 20% of the
rice paddy produced is husk, annualy creating 120 million tones of total production
(Giddel and Jivan, 2007). The outer part of rice husk structured by dentate
rectangular elements, which are generally composed of silica coated with a thick
cuticle and surface hairs. The inside part and inner epidermis are contain of silica.
Besides in agricultural side, rice husk can be applied in industrial processes as well.
The applications are including steel foundries, for houses manufacture material and
refractory bricks (Bronzeoak, 2003).

8


Rice husk typical analysis shown in Table 1. The composition of each property
is depends on rice variety, soil content, climate altitude, as well as geographical
localized of the cultivation (Agus, 2002).

Table 3. Rice Husk Property
Property
Bulk density (kg/m3)
Hardness (Mohr’s scale)
Ash (%)
Carbon (%)
Hydrogen (%)
Oxygen (%)
Nitrogen (%)
Sulphur (%)
Moisture

Range
96-160
5-6
22-29
≈ 35
4-5
31-37
0.23-0.32
0.04-0.08
8-9

(Source: Muthadhi and Kothandaraman, 2007)
Considering to the rice husk characteristic properties and chemical
composition, it has been found about the application of rice husk on the side of
construction, energy production, production of various chemical product, and et
cetera (Valchev et al., 2009).
Burning process towards rice husk at high temperature will convert rice husk
to another form which is rice husk ash that contains silica. Rice husk ash does not

only provide sufficient benefits, but it also has significant market value. It has been
demonstrated that 85% to 95% amorphous silica can be converted from it
(Krishnarao and Subramanian, 2001).
2.3.

Natural Adsorbent
Adsorbent is a solid substance that has an ability to adsorb other subtances

from one to another surface without any covalent bonding. Example of natural
adsorbents are activated charcoal, rice husk, penaut shell, and et cetera (Miller,
2003). Several studies have been showed the ability of aqueous solutions such as
orange and apple residue, rice husk, peanut and coconut shell, as well as tea are
effective in removal toxic substances (Alagumuthu and Rajan, 2010). Several low
cost adsorbent resulting from natural and and anthropogenic sources have been
investigated for treatment of waste water contaminated by heavy metals. Generally,

9


adsorbent used are natural materials from agricultural waste, industrial byroproducts
as well as modified biopolymers (Tripathi, 2015).
2.3.1. Rice Husk as Adsorbent
Rice husk contains large amount of hydrocarbon in the form od cellulose and
lignin content. Those can be aplied as a raw material to make activated carbons
which have a complex porous structure. There are two methods in term of rice husk
activation: physical (thermal) activation and chemical activation. Afterwards,
carbonization and activation usually done in a single step by using a chemical agent
while physical activation produces activated carbon and it offers low specific area.
Activated carbon and or adsorbent from rice husk was investigated effective in term
of adsorption process considering its microporous structure (Cristina and Rosa,

2008).
Andi (2010) has been reported the optimum time of rice husk in absorbing
Pb2+ ion is approximately 85 minutes the optimum contact time for rice husk in
absorbing Pb2+ ions is 85 minutes with an absorption capacity of 0.232 mg/g. In
coorelated by the longer contact time of adsorption process, the higher Pb2+ ion will
be absorbed. Rice husk has an ability of absorption due to its large pores as well as
surface area on the surface of rice husk.
Rice husk is composed by 80% of organic matter as the main element which
are cellulose compounds and lignin as functional group as hydroxyl (OH), carboxyl (COOH), ketones (R-O-R) that will react to bind heavy metals (Dupont, 2005).
2.4.

Test Species – Oreochromis niloticus
Scientific Classification
Kingdom

: Animalia

Phylum

: Chordata

Class

: Actinopterygil

Order

: Perciformes

Family

: Cichlidae

Genus

: Oreochromis

Species

: O. Niloticus

10


Nile tilapia (O. niloticus) was one from the early fish species cultured.
From illustrations by Egyptian crypts that commend if nile tilapia were cultured
over and above 3000 years ago. Tilapia have been knew as "Sain't Peter's fish"
in resource to biblical quotation about the fish fed to the large group. In africa,
Nile tilapia still be the most largely cultured species. Nile tilapia fish has
adaptation ability to the water temperature lower than 10 to 11°C (Thomas and
Michael, 1999).
2.5.

Histopathological Effects
Patnaik et al. (2010), were evaluating histopathological changes on the
gills of Cyprinus carpio communis exposed to different concentrations of lead
and cadmium. The results of the damage by Lead were fusion and disintegration
of primary lamellae as well as extensive vacuolization upon disruption of
epithelial lining. On the other side, hyperplasia, congestion, and vaculization
were recorded due to the effect of the Cadmium.

The effect of heavy metal, was examined on gill morphology of fresh
water fish from Garmat Ali River adjacent to Al- Najebyia Power Station Iraq.
Morphological gill changes were identified by the adhesion of some secondary
lamellae correlated to the imflammatory cells, congestion, and loss of epithelial
lining made cappilaries were the only ones left. Fragment of secondary lamellae
were detected due to the destruction of structures affected by the heavy metals.
In the study by Poleksic et al. (2009), the Sterlet (Acipenser ruthenus L.) were
indicated by some heavy metals (Cd, As, Pb, Cr, Hg, Cu, Ni, Fe, Mn, and Zn).
The histopathological study was conducted and showed 95% of structural
changes comprised by the loss of the secondary lamellae and hyperplasia of the
respiratory epithelium.
Abalaka (2015) investigated the effect of heavy metals concentration in
Tiga dam, Nigeria using Auchenoglanis occidentalis as the indicator. Their
bioaccumulation and histopathologic alterations on the fish gills were carried
out. The present of some metals were detected as zinc (Zn), cadmium (Cd), lead
(Pb) and iron (Fe). Morphological changes showed that the the toxic effect of
Pb and Cd affected to the damage of membrane integrity with subsequent loss
11


of membrane-bound enzyme activity arising in cellular death. The alterations
changes comprised of hyperplasia and the detachment of the gill epithelial cells
followed by the fusion of lamellae caused by the effect toxic contaminations.

12


PART III. METHODOLOGY
3.1.

Place and Time
This experiment started from May 2017 until September 2017. Toxicity test and

heavy metal adsorption test using fish as indicator were conducted at Aquaculture
Laboratory, Faculty of Agriculture, Sriwijaya University, Inderalaya Campus.
Adsorbent adsorption examination was conducted at Fish Product Technology
Laboratory, Faculty of Agriculture, Sriwijaya University, Inderalaya Campus.
Histophatological examination was conducted at Laboratory of Anatomical Pathology
Laboratorium Barokah Palembang.
3.2.

Equipment and Materials
Equipment and materials used for this study are listed as in Table 2 and Table 3

below.
Table 4. List of Equipment Used
Toxicity Testing










Aquarium
Aerator
Fish strainer

Erlenmeyer
Beaker
Spatula
Tweezers
Gloves
Mask

Bioadsorbent
Histopathological Testing
Testing
Apparatus used in
 Aquarium
laboratory :
 Aerator
 Graduated cylinder
 Erlenmeyer
 Glass funnel
 Pipettes
 Cassettes
 Beaker
 Dissecting Knife
 Shaker
 Scissors
 Analytical
 Scalpel
balance
 Rubber gloves
 Magnetic
funnels
 Aprons

 Gloves
 Mask
 AAS (Atomic
 Microscope slides
Absorption
Spectroscopy) Instruments used in
laboratory:
 Histokinet
 Shandon histocentre 3
 Shandon finesse 325
(microtome)
 LAB Line Barnstead
26025 Slide Warmer
 Tissue floating bath
 OLYMPUS BX51
microscope

Additional
 Pen
 Pencil
 Book
 Plastic
bag

13


Table 5. List of Material Used
Toxicity
Testing

 Fish
(OreochromisNiloticus)
 FeSO4 pro analis (PA)

3.3.

Bioadsorbent
Testing
 Rice Husk
 Aquadest
 whatman 42 filter
papper












Histopathological
Testing
Formaldehyde solution pH 7.0
Ethanol
Alcohol
Xylene

Acetone
Paraffin wax
Haematoxylin
Eosin
Dibutyl
PhthalateXylene

Fish preparation, Preparation of Fe2+ Solution and Adsorbent Preparation

3.3.1. Fish Preparation
Black tilapia fish obtained from Balai Benih Ikan Indralaya, Ogan Ilir. Fish
were selected based on the size 10–12cm. Size of the fish was considered to avoid
errors due to difference striking size of fish will show different result to the
treatment. Selected fish were the fish that swim actively, not sick, and no defects.
They were acclimated to laboratory conditions for 7 days prior to adapt selected fish
to the experiments (Edy, 2001).
3.3.2. Preparation of Fe2+ Solution
Stock solution of Fe2+ ion was made by weighing 2.7g of FeSO4 pro analyst
(Days, 2015). Using beaker glass, added 1000mL of aquades and stirred well with
magnetic stirrer. This stock solution is used to make a fixed amout of Fe solution
with concentration 4mg/L, 8mg/L, and 10mg/L that used for preliminary toxicity
test using diluted formula:
V 1 x C 1 = V2 x C 2
Where :
V1
= Volume of Fe2+ stock solution needed to make the new solution
C1
= Concentration of Fe2+ stock solution
V2
= Final volume of new Fe2+ solution

C2
= Final concentration of new Fe2+ solution
3.3.3. Adsorbent Preparation
The rice husk were initially washed with water and separated from other dirt.
After this, the cleaned and dried husk was burned at 250°C for 2.5 hours. After
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cooling, the rice husk ash was lightly sieved through 212μm mesh. Finally, the ash
fraction retained on the mesh was stored and it was used as the adsorbent in heavy
metal adsorption process (Nurhasni, Hendrawati and Nubzah, 2014).
3.4.

Methods

3.4.1. Toxicity Experiment
Preliminary toxicity experiments were run to detemine LC50 of iron (II)-sulfate
(FeSO4) in O. niloticus. During toxicity experiments, there were four aquariums
containing 10L of water and 10 fish was chosen randomly for each aquarium. The
iron (II)-sulfate (FeSO4) were exposed to the three aquariums and one aquarium as a
control that not exposed to the test chemical. The concentrations used for toxicity
experiments were 4mg/L, 8mg/L and 10mg/L which considered as test solutions.
During toxicity experiments, fish were not fed. The LC50 values evaluated by using
probit analysis (SPSS).
3.4.2. Heavy Metal Adsorption Test using Fish as Indicator
This part was rather mostly aimed at the adsorbent potentials of rice husk ash
on removal of heavy metals (Fe2+) in the water. The experimental procedures were
carried out in a glass aquaria which consist of 10L water and ion Fe mixtured. The
control solution (C) obtained from preliminary toxicity tests (LC50). Batch
experiments were varied into C, C-20%, C-10%, C+10%, C+20% mg/L. Each glass

aquaria that contained 10L of various FeSO4 water, inserted with 40 grams of rice
husk concentrations. This was run for 24 hours and separated the rice husk ash from
the water when it finished (Jubaedah, 2017)*
In addition to see the result of adsorbent experiment, 10 fish were chosen
randomly for each aquarium. In this case, there are six treatments and five
replications. During that time, the dead fish were carefully taken out from aquarium
and removed the portions of gills for histopathological examination.
3.4.3. Adsorbent Adsorption Examination (Qaiser, Saleemi and Ahmad, 2007;
Prasad et al, 2008)
This part aimed to investigates the adsorption of heavy metal (Fe) onto rice
husk ash. 1 grams of rice husk ash were weighed into 250mL erlenmeyer. 100mL of
*

Personal Communication

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the solution contains 0.87mL of ion FeSO4 were mixed to the adsorbent. Erlenmeyer
was covered up with aluminium foil and shaken using shaker 200rpm for 30, 60, 90,
120 and 150 minutes. After this, adsorbent mixture were separated from the solution
with using whatman 42 filter paper. The final filtrate product from adsorbent
adsorption of Fe solution was analyzed using AAS (Atomic Absorption
Spectroscopy).
3.4.4. Histopathology Examination
Histopathological examination was conducted by technicians in the
laboratory. Procedures required include these following steps: (Raphael, 1976;
Bancroft and Gamble, 2002)
Fixation
Fixation processes started after gill portion were taken out and soaked onto 10%

neutral buffered formaldehyde solution with normal pH for 24 hours or more. Volume
of formalin is 1-10 of tissues volume.
Tissue Processing
Tissue processing started by tissue dehydrated or removal of water and formalin
replacement with a series of sequential alcohols 50%, 70%, 95% dan 100%. The next
step was clearing process to remove alcohol content using Xylene for one hour,
continued with tissue infiltrated using paraffin. After this, embedding processed were
conducted by taking the form of paraffin block and placed tissue cassette in melted
paraffin. Mold was filled with hot paraffin, then colled down the tissue while it
finished.
Sectioning
In sectioning processes, microtome were used to cut off the paraffin block at
5µm thickness. The ribbon of sections were taken on a hot water bath and the floated
tissue were picked up onto slides. Slides were placed and dried on a hot plate at about
50°C for 30 minutes. It was important to make a flat slide, no air bubbles, no stretch
or breaks.
Staining procedure using Haematoxylin and Eosin
During this step, the slide were placed and immersed to deparaffinize and
hydrate in xylene to dissolve paraffin using various alcohol 95%, 90%, 80%, 70%,
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50% and 30% for 5 minutes each one, and it rinsed in running tap water for 5 minutes.
After that, slides were stained by using haematoxylin for 15 second to 10 minutes
depending on the age of the eosin and the depth of the counterstain desired. The next
step was removing the excess eosin in various absolute alcohols 30%, 50%, 70%,
80%, 90% and 95% in 3 minutes for each of the slide. While dehydrated in 95% and
then 100%, the slides were soaked for 2 to 3 times of 2 minutes until the excess eosin
well removed. After the whole process finished, the slides were dried overnight and
ready to be examined using microscope.

Experimental Chart
START

PREPARATION




TOXICITY TEST
Concentration
(0mg/L, 4mg/L, 8mg/L, 10mg/L)
Optimum Concentration (LC50)

HEAVY METAL ADSORPTION TEST
USING FISH AS INDICATOR
 Solution variation
- C with adsorbent treatment
- C without adsorbent treatment
- C-10% with adsorbent treatment
- C-20% with adsorbent treatment
- C+10% with adsorbent treatment
- C+20% with adsorbent treatment
 Rice husk ash (40g)
 Adsorption duration (24h)
 Indicator (10 fish)
 6 treatments, 5 replication

Equipment and
Material Preparation
Fish Preparation

Preparation of Fe2+ Ion
Solution

ADSORBENT ADSORPTION
EXAMINATION
 Rice husk ash (1g)
 Erlenmeyer (250 mL)
 Distilled water (150 mL)
 FeSO4 (0.87 mL)
 Speed/duration (200rpm/
30, 60, 90, 120, 150
minute)
 AAS

Heavy Metal Reduction
HISTOPATHOLOGY EXAMINATION
 Fixation
 Tissue Processing
 Sectioning
 Staining using H&E

FINISH

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PART IV. RESULTS AND DISCUSSIONS
This part showed the results obtained from each experimental that have been
conducted. The results are consisted of preliminary toxicity test, indicator heavy
metal adsorption test, adsorbent adsorption examination and histopathology

observation of fish gills.
4.1. Preliminary Toxicity Test
Preliminary toxicity tests were aimed to determine LC50 (Lethal Consentration)
of iron (II)- sulfate (FeSO4) in O. niloticus. There were 4 aquariums containing 10L of
water and 10 fish were chosen randomly for each aquarium. Three aquariums were
exposed to the iron (II)- sulfate (FeSO4) and one aquarium as a control was not
exposed to the test chemical.The concentrations used for toxicity testing were 4mg/L,
8mg/L and 10mg/L which were considered as test solutions. This experiment was run
for approximately 24 hour. From preliminary toxicity tests, the data was collected
shown at the table below.
Table 6. Preliminary Toxicity Test Result
Concentration
0mg/L (Control)
4mg/L
8mg/L
10mg/L

Population
10
10
10
10

Duration
24 hours
24 hours
24 hours
24 hours

Mortality

1
4
7
9

From observations, the highest number of fish mortality was found at the
highest concentration of 10mg/L. At concentrations of 4mg/L, the number of fish
mortality was four while at a concentration of 8mg/L was seven. The highest number
of fish mortality that occurred at the treatment of 10mg/L of iron (II) sulfate is
expected because 10mg/L was the biggest number of concentration that given to the
aquarium. The higher number of concentration given, the bigger number of harmful
solution that enters to the fish. Fish responds to the pollutant that enters to the water by
moving faster in order to avoid contaminated water. Decreasing frequency in fish
movement is resulted from the energy of the fish has decreased. Decreasing energy is
caused by the damage of mitochondria where it is one part of cell organelles that has
function to produce energy through the process of aerobic respiration and regulate cell
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