Peanut shell as natural adsorbent of wastewater treatment containing copper II sulfate (CuSO4) and its toxicological effects on oreochromis niloticus

The present study has been conducted to determine peanut shell as natural adsorbent of wastewater treatment containing copper-II-sulfate CuSO4 and its toxicological effects on Nile Tilap

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

RIZKY GUSTIANI VIDYA

PEANUT SHELL AS NATURAL ADSORBENT OF WASTEWATER TREATMENT CONTAINING COPPER-(II)-SULFATE ( CuSO 4 ) AND ITS

TOXICOLOGICAL EFFECTS ON OREOCHROMIS NILOTICUS

BACHELOR THESIS

Study Mode : Full-time

Major : Environmental Science and Management Faculty : Advanced Education Program

Thai Nguyen, 23/09/2019

Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name Rizky Gustiani Vidya

Student ID DTN1454290074

Thesis Title Peanut Shell as Natural Adsorbent of Wastewater

Treatment Containing Copper-II- Sulfate (CuSO4) and

Its Toxicological Effects on Oreochromis niloticus

Supervisor (s) Prof Nguyen The Hung

Dr Ho Ngoc Son Supervisors’

Signatures

Abstract:

High levels of water pollution can destroy aquatic life Pollutants are taken up

by plants and animals and can enter the human body, resulting in many health problems The situation is worse in countries where people do not have access

to potable water, and in many instances polluted water is used as a source of drinking water The present study has been conducted to determine peanut shell as natural adsorbent of wastewater treatment containing copper-(II)-sulfate (CuSO4) and its toxicological effects on Nile Tilapia (Oreochromis niloticus) This study reported that the removal of copper-(II)- sulfate (CuSO4) from aqueous solution could be using pure and physically pretreated biomass

from Arachis hypogea (peanut shell) which this material was indigenous,

easily available, surplus by-products for biosorption studies The preliminary toxicity test was carried out with 5 different concentration treatments to determine LC50 and it found to be 21.91 mg/L Adsorbent experiments used fish as bioindicators of heavy metal adsorption using 5 concentration variations with 6 treatments and 5 replications The used concentrations of CuSO4 are 21.91 mg/L without adsorbent; 17.53 mg/L with adsorbent; 19.72 mg/L with adsorbent; 21.91 mg/L with adsorbent; 24.10 mg/L with adsorbent; 26.29 mg/L with adsorbent The results showed that Nile Tilapia mortality was still found in treatments using adsorbents accompanied by changes in fish behavior such as loss of equilibrium, change in body color, irregular

swimming activity, rapid jerk movement and aggressiveness before the fish died The adsorption capacity of peanut shell for adsorbent adsorption examination was effective during the contact time between pollutants and adsorbents and the absorption capacity occurred The observation of histopathological changes was also conducted on gill tissue using Hematoxylin and Eosin Staining Methods The observed changes in gills were congestion, curling bend of secondary lamellae, epithelial proliferation of secondary lamellae, epithelial rupture of secondary lamellae, fusion of secondary lamellae, and telangiectasia Thus, this study provided that peanut shell as natural adsorbents was effective to absorb water that had high content of copper

Keywords:

Water pollution, peanut shell, Copper, Cu, Copper-(II)-Sulfate, CuSO 4 , natural adsorbent, histopathology, Oreochromis niloticus, Nile Tilapia, toxicity, gills

Number of pages: 64

Date of Submission: September 23rd, 2019

ACKNOWLEDGEMENT Alhamdulillah, I praise and thank Allah SWT for giving me the strength,

knowledge, ability and opportunity to complete this research study and to persevere and accomplish it satisfactorily From bottom of my heart, I would like to express my deepest appreciation to all those who provided me the opportunity to complete this research

First and foremost, I would like to express my sincere gratitude and deep

regards to my supervisor: Assoc Prof Dr Nguyen The Hung of Thai Nguyen

University of Agriculture and Forestry who guided me wholeheartedly when I

implemented this Also, I want to express my thanks to Dr Ho Ngoc Son, the

second supervisor, for his supervision, encouragement, advice, and guidance in writing this thesis

Besides my supervisors, I would like to thank Dr.-phil Arinafril of Sriwijaya University, Indralaya, Indonesia and Ph.D Dr Krisna MURTI in

Department of Anatomical Pathology, who kindly assisted me with the histopathological detection in this dissertation and was very patient with my knowledge gaps In addition, formal thanks should be offered to the Rector of

Sriwijaya University, Prof Dr Ir H Anis Saggaf, MSCE, for granting my

internship acceptance I would also like to acknowledge with much appreciation

to the Dean of Faculty of Agriculture in Sriwijaya University, Prof Dr Ir Andy Mulyana, M Sc., who gave the permission to conduct my research in

Faculty of Agriculture, Sriwijaya University

Special thanks to Mochamad Syaifudin, S Pi., M Si., Dade Jubaedah,

S Pi., M Si., and Dr Mohammad Amin, S Pi., M Si., from Budidaya

Perairan, Faculty of Aquaculture, Sriwijaya University

My sincere thanks also go to Mrs Nurhayani, Mrs Ana, Ms Naomi, other staffs and friends in Laboratory of Budidaya Perairan and Laboratory of

Teknologi Hasil Perikanan, Sriwijaya University for helping and providing me necessary equipment as well as knowledge for fish anatomy

My parents, Mohammad Burhanuddin and Yuliani, deserve special

mention for their inseparable support and prayers

Finally, special thanks to my friends for their love and moral support throughout my study and I would like to thank everybody who was important

to the successful realization of this thesis, as well as expressing my apology that

I could not mention personally one by one

Thai Nguyen, September 23 rd , 2019

Future Environmentalist, Rizky Gustiani Vidya

TABLE OF CONTENT

Acknowledgement iii

TABLE OF CONTENT v

List of Figures 1

List of Tables 3

PART I INTRODUCTION 4

1.1 Background and Rationale 4

1.2 Research’s Objectives 7

1.3 Research Questions and Hypotheses 7

1.4 Limitations 8

PART II LITERATURE REVIEW 9

2.1 Copper 9

2.1.1 Copper-(II)-Sulfate 10

2.1.2 Toxic Effects of Copper-(II)-sulfate 13

2.2 Low-cost Adsorbent 13

2.2.1 Peanut Shell 15

2.3 Histopathological Effects .17

2.4 Test Species .19

PART III METHODS 21

3.1 Place and Time 21

3.2 Materials & Equipment 21

3.2.1 Toxicity Testing 21

3.2.2 Adsorbent Testing 21

3.2.3 Histopathology Examination 22

3.3 Preparation 23

3.3.1 Fish Preparation 23

3.3.2 Preparation of Cu2+ Solution 23

3.3.3 Adsorbent Preparation 24

3.4 Methods 24

3.4.1 Toxicity Testing 24

3.4.2 Adsorbent Experiment Using Fish as Bioindicator 24

3.4.3 Adsorbent Adsorption Examination 25

3.5 Histopathological Examination 26

3.5.1 Fixation 26

3.5.2 Tissue Processing 26

3.5.3 Sectioning 26

3.5.4 Staining procedure using Hematoxylin and Eosin 27

PART IV RESULTS AND DISCUSSION 28

4.1 Preliminary Toxicity Test 28

4.2 Adsorbent Experiment Using Fish as Bioindicator 30

4.3 Adsorbent Adsorption Examination 34

4.4 Histopathological Observation of Gills 36

PART V CONCLUSIONS .52

REFERENCES 54

APPENDICES 65

LIST OF FIGURES

Figure 1 Effect of Contact Time on the Absorption of Cu2+ Ion (mg/L) 35 Figure 2 Normal histological structure of gills 37 Figure 3 Copper-(II)-Sulfate (CuSO4) with concentration of 21.91 mg/L without adsorbent treatment Congestion (Cs); Epithelial proliferation of secondary lamellae (EP); Fusion of secondary lamellae (F); Epithelial rupture of secondary lamellae (ER); Telangiectasia (Tel); Curling bend of secondary lamellae (CB) H&E, magnification x100 .38 Figure 4 Tilapia gills exposed to 17.53 mg/L Copper-(II)-Sulfate (CuSO4) with adsorbent treatment (4a); Tilapia gills evidencing fusion of secondary lamella (4b); Tilapia gills presenting curling bend of secondary lamella (4c); Tilapia gills showing epithelial proliferation of secondary lamellae in which secondary lamellae cells grow excessively (4d); A-D are stained with H&E, magnification of

A is 100x while B, C, and D is 400x .41 Figure 5 A) Copper-(II)-Sulfate (CuSO4) with concentration of 19.72 mg/L with adsorbent treatment H&E, magnification x100; B) Tilapia gills (O niloticus) showing congestion and fusion of secondary lamellae H&E, magnification x400; C) Tilapia gills evidencing epithelial proliferation of secondary lamellae H&E, magnification x400 .42 Figure 6 A) Copper-(II)-Sulfate (CuSO4) with concentration of 21.91 mg/L with adsorbent treatment H&E, magnification x100; B) Tilapia gills presenting telangiectasia H&E, magnification x400; C) Tilapia gills showing disintegration

of secondary lamellae H&E, magnification x400 .43 Figure 7 Tilapia gills exposed to 24.10 mg/L Copper-(II)-Sulfate (CuSO4) with adsorbent treatment (7a); Tilapia gills showing epithelial proliferation of secondary lamellae (7b); Tilapia gills evidencing fusion of secondary lamella (7c); Tilapia gills showing curling bend of secondary lamella (7d); A-D are stained with H&E, magnification of A is 100x while B, C, and D is 400x .45 Figure 8 A) Copper-(II)-Sulfate (CuSO4) with concentration of 26.29 mg/L with

bend of secondary lamellae and congestion H&E, magnification x400; C) Tilapia gills showing fusion of secondary lamellae H&E, magnification x400; D) Tilapia gills presenting epithelial proliferation of secondary lamellae H&E, magnification x400 47

LIST OF TABLES

Table 1 Physical and Chemical of Copper-(II)-Sulfate Material 12

Table 2 The Toxic Effects of Copper-(II)-sulfate 13

Table 3 Types of Adsorbents Based on Surface Area 14

Table 4 Adsorption Capacities for Low-Cost Adsorbents 15

Table 5 Chemical Composition of Peanut Shell 16

Table 6 Preliminary Toxicity Test Result 28

Table 7 The Value of Varied Concentrations 30

Table 8 Effect of CuSO4 (Copper-(II)-sulfate) on Nile Tilapia in Five Replications .31

Table 9 Effect of Copper-(II)-sulfate (CuSO4) to The Behaviour of Nile Tilapia in 24 Hours 32

Table 10 Effect of Contact Time with The Adsorbent Solution of Cu2+ Ion 35

Table 11 Gills Alterations 50

CHAPTER I INTRODUCTION

1.1 Background and Rationale

The industrial development has progressed very rapidly The increasing number of industries not only has a positive impact but also has a negative impact, for example as a result of the industrialization process, industrial effluents are produced in the form of liquid, solid, and gas which cause environmental pollution Liquid waste in this industry contribute to the release of toxic heavy metals in the water flow The impact of this industrial waste results in an increase in the amount

of toxins and heavy metal as pollutants in the environment Heavy metals, such as

Hg, Pb, Cr, Ni, Cu, Cd and Zn, can accumulate in organisms and in large quantities

it can cause poisoning when absorbed in the body (Ngah et al., 2004)

One of major issue throughout the globe is heavy metal pollution that caused by industrial wastewater Exceptionally, copper (II) ions, whose indicates the adverse impacts on human and aquatic life considered as dangerous, and by all means are not environmentally-safe As a result, they have to be disposed from effluents in order to obtain quality standards for the environment According to U.S Environmental Protection Agency (EPA), the allowed restriction for copper

in drinking water is 1.3 mg/L and the estimated intakes of copper in the general population are 0.15 mg/day from drinking water, and approximately 2 mg/day from food (US EPA, 2007) Copper is a commonly used material that can be discovered to be a contaminant in food Contamination occurs because of using copper material for food packaging, notwithstanding the essential nature of copper,

cancer for workers that expose to copper due to continuous inhalation of containing spray Copper is discharged into the atmosphere in variety methods; it finds its way into water-streams leading to environmental contamination that has threat to every organism This may cause significant and complex issues

copper-(Papandreou, Stournaras & Panias, 2007; Pentari et al., 2009)

Water contamination caused by various pollutants has been a big concern which it has had an adverse effect on natural aquatic systems for decades this occurs due to the release of heavy metals by industry and other man-made activities that cause contamination resulting in a degradation in water quality The ecological balance of the recipient and the diversity of aquatic organisms may be adversely affected by heavy metal contamination These aquatic organisms can nevertheless

be widely used to biologically monitor environmental deviations from anthropogenic contaminants and they are important instruments for identifying potential environmental problems before a system's health is critically modified or endangered Among aquatic species, fish is one of inhabitants that cannot avoid

the detrimental effects of the pollutants (Farkas et al., 2003; Figueiredo-Fernandes

et al., 2007) Fish are used in general for the health evaluation of aquatic

ecosystems by considering their change in physiology Moreover, they act as biomarkers of environmental pollution and the organs of aquatic animals can accumulate copper when they are exposed to different toxic concentrations (Mazon

et al., 2002) Copper may result in redox reactions which produce free radicals and

thus can cause biochemical and morphological changes Fish gill is well-known as the most commonly organ to response to pollutants in environment In addition,

gills are the first place of waterborne pollutants due to it is the main location for copper uptake, and it has a direct and constant contact with the outside

environment (Figueiredo-Fernandes et al., 2007)

Adsorption is the most efficient technique used for purifying and recovering

Cu (II) ions from waste due to high efficiency and easy handling (Kurniawan et al., 2006) In addition, cost consideration is one of alternative ways that need to be considered to determine what kind of the technology will be used for processing wastewater containing heavy metal such as Cu because cost is an important parameter in selecting adsorbents In general, the adsorbent can be said as cheap if

it only requires a little process, the material is abundant and it is a by-product of waste from industries (Arifin, 2003) The use of peanut shell as biomaterials from agricultural waste as a substitute for activated carbon or ion exchange resins to absorb toxic compounds has begun to be examined The components of peanut shell are expected to be used as heavy metal adsorbents, which are cellulose that found on peanut shell cell walls Cellulose has an active OH group which is capable

of binding heavy metals (Windasari, 2009)

Furthermore, to deal with problems related to pollutions, this study is an essential instrument which can be used to investigate the impact of human activities and consider the adverse effects that can occur not only on public health but also on the environment The aim of this study is to determine the effectiveness

of peanut shell as a natural adsorbent in treating wastewater containing copper-

(II)-sulfate by looking at the toxic effects on Oreochromis niloticus.To determine more about how much copper-(II)-sulfate can be absorbed by peanut shell as a

natural adsorbent, adsorbent adsorption examination is conducted and followed by histopathological examination on fish gills to evaluate the damage and histopathological alterations which is caused by CuSO4

1.2 Research’s Objectives

In this context, the aim of this study is to investigate the treatment of

wastewater using peanut shell and its toxicity effects on Oreochromis niloticus

at different concentrations of Copper-(II)-Sulfate likewise to reveal the histopathological alterations on its gills

1.3 Research Questions and Hypotheses

- HA (Alternative Hypothesis): There is effectiveness of using peanut shell

to adsorb copper-(II)-sulfate (CuSO4)

⁻ Experimental results on the histopathological effects on the gills tissue

of Nile Tilapia in literature which caused by CuSO4 are difficult to find

CHAPTER II LITERATURE REVIEW

2.1 Copper

Copper is a rudimentary metal with a cubic, crystalline facial structure and

it is an element that is numerous in nature Due to the band structure, it reflects light and absorbs other frequencies in the visible spectrum, so it has pleasantly reddish color Rocks, soil, water, sediment, and, at low level air are where copper

is shaped and there are still around 50 parts of copper for every million pieces of soil (ppm) or, in different ways, 50 grams of copper for every 1,000,000 grams

of soil (ATSDR, 2004) Copper is a necessary element for all biological organisms, from microorganism cells to human because it has a role to make red blood cells, control the nerve cells as well as the immune system Depending on the source of the biological material, Cu content ranges from parts per billion to parts per million however, due to human activities large amounts of Cu have been released into the environment which adversely affect all of organisms During the past five decades, about 939,000 tons of Cu were released into the environment

around the world (Singh et al., 2003)

The increasing levels of copper make it toxic to mammals (including humans) and also other organisms which will disrupt the regulatory systems such

as supply, distribution, storage and excretion Cu is an essential feature for all organisms in the aquatic environment to continue to live However, there are many sources of Cu that derived from human activities or better known as anthropogenic activity, such as industrial processes, electrical wiring, smelters,

Hornberger, 2002; Nordberg et al., 2007) Different kind of industries,

agriculture and municipal sources emit many pollutants As a result, it permanently contaminates water which can transfer to the food chain (Vukovic

et al., 2011)

Copper speciation, solubility and complexation are important factors that influence Cu's toxicity in the aquatic environment The number of dissolved copper relies heavily on the pH of the water The relation between this heavy metals can change the toxic effects both on positive and negative for aquatic organisms (Jezierska, Ługowska & Witeska, 2001) Copper can be found easily

in water due to natural sources and anthropogenic causes Moreover, copper is also one of the leading pollutants which affect water, soil, and air quality Especially, copper can create permanent affects to living organisms that participate in the food chain which can cause many diseases, including cancer

(Doong et al., 2008; Zhang et al., 2010)

2.1.1 Copper-(II)-Sulfate

Copper-(II)-sulfate is commonly referred to "blue stone" because it occurs

in nature as large blue crystals It is used for agricultural purposes such as fungicide, algaecide, and nutritional supplement Gardeners and commercial farmers usually apply copper-(II)-sulfate to prevent problems with fungus or mold and of there are negative and positive effects of using copper-(II)-sulfate on plants For example, the status of the soil, the frequency of application and the quantity of copper-(II)-sulfate will decide the effects of copper-(II)-sulfate on plants (Allen, 2016)

Due to its high solubility in water, disproportionate amounts of (II)-sulfate should be prohibited of lakes, streams, and ponds Water contamination can occur due to the inappropriate cleaning of application equipment or disposal of waste which is associated with this material (US EPA, 2012) A rise in agricultural production has led to an increased in the impacts on freshwater systems caused by wastewater releases CuSO4 is used extensively as

copper-a fungicide in copper-agriculture When it is used excessively, high concentrcopper-ation of fungicides will be detected in several aquatic ecosystems such as freshwater and groundwater The disposal of industrial effluents aggravates this situation

US EPA, 2012)

The following of Physical and Chemical of Copper-(II)-Sulfate material showed in Table 1:

Table 1 Physical and Chemical of Copper-(II)-Sulfate Material

Synonym(s)

Copper-(II)-Sulfate; Cupric sulfate; Copper sulphate; Cupric sulfate anhydrous;blue stone; blue vitriol; cupric sulphate; Roman vitriol; Salzburg vitriol; blue copperas

Formula CuSO4 or CuO4S

CAS Registry Number 7758-98-7

Molecular Weight 159.602 g/mol

Molecular weight 159.61

Decomposes at 560 Specific gravity (20/4

Soluble in methanol; Slightly soluble in ethanol

Conversion factors at

25°C ppm to mg/m3

Since these substances exist in the atmosphere in the particulate state, the concentration is expressed as mg/m3

(Source: ATSDR, 2004)

2.1.2 Toxic Effects of Copper-(II)-sulfate

Table 2 The toxic effects of Copper-(II)-sulfate

Dermal Mild irritant skin A repetitive implementation to

fractured skin can lead in systemic copper uptake Ocular

Eye irritation, corneal necrosis and opacity may blead to the presence of crystals in the conjunctival sac

Ingestion Very low consumption (in milligrams) is probable to

result nausea and vomiting

Moderate/substantial

ingestions

The abdominal pain and diarrhea follow in minutes with nausea, vomiting and also metallic taste The color of secretions becomes blue/green Severe swelling of the gastrointestinal tract may lead to hematemesis and/or hypoglycemia Severe intoxication involves growth and intravascular haemolytic disease (generally 24-48 hours after toxicity), metabolic and obstructive liver damage The description of this disease includes methaemoglobinemia, coma, convulsions, rhabdomyolysis, muscle failure and cardiac arrhythmia The risk of gastric aspiration in obtunded patients is high

Inhalation

It is reported for acute occupational inhalation of fungicides containing copper sulphate, which have progressive dyspnea, cough, wheeze, myalgia, malaise, micronodular, reticular opacity on the chest X-ray (which may coalesce) and a lung defective Additional characteristics include copper-containing hepatic granulomas and hypergammaglobulinemia

(source: ATSDR, 2004)

2.2 Low-cost Adsorbent

Adsorbent is a material which can withstand certain fluid stage elements and most adsorbents are extremely porous substances Adsorption takes place primarily on the pore walls or at certain locations within the particles (Rahmayani

& Siswarni, 2013) Five types of adsorbents based on surface area can be seen in Table 3

Table 3 Types of Adsorbents Based on Surface Area

Activated Carbon

a microcrystalline material made by thermal decomposition of wood, plants, shell, coal, etc Has an area of 300-1200

m2/g with an average pore diameter of

10 to 60 A

area of 600-800 m2/g with an average pore diameter of 20-50

Activated Alumina has a surface area of 200-500 m

2/g with

an average pore of 20-140 A

Molecular Sieve Zeolite

used to separate hydrocarbons and mixtures thereof Has a pore size of 3-

10 A Synthetic Polymers or resins used to absorb non-polar organic

compounds

(Source: Davis & Cornwell, 2013)

A low-cost adsorbent is determined to be plentiful in nature or to be a product of or waste in another industry To remove heavy metal in wastewater, activated carbon is most commonly used as adsorbent because of its great capacity

by-to adsorb Even though it is a chosen sorbent, its extensive use is limited because

of the high cost For diminishing the cost of treatment, trials have been made to find economical alternative adsorbents also known as low-cost adsorbents (Aydin, Bulut & Yerlikaya, 2008) The waste can be used as cost-effective adsorbents which can be used as water purification, helping industries to reduce the cost of waste disposal therefore, it also gives a potential to agricultural waste as natural adsorbent Using low-cost adsorbent for the removal of heavy metals is looked to

be more encouraging in the long terms as agricultural wastes or industrial products exist locally and extensively as natural materials which can be utilized as

by-low-cost adsorbents (Nur AA et al., 2013) Table 4 listed examples of low-cot

adsorbent as wastewater treatment

Table 4 Adsorption Capacities for Low-Cost Adsorbents

Low-Cost

Adsorbent

Heavy Metal

Adsorption Capacities

(Ofomaja, Unuabonah and

(Aydin, Bulut & Yerlikaya, 2008)

Peanut or groundnut (Arachis hypogaea L.), is a plant originating from

the Americas, precisely from the region of Brazil (South America) Peanuts are short-growing crops that grown in terrain with altitude 0-500 meters above sea level Peanut is the second most important crop in the world after soy beans because it supplies food for human and livestock and it is also form valuable dietary protein component in the absence of meat Since peanut shell are abundant in nature, economical, need little processing and also are effective materials, they can be considered as low-cost adsorbents (Sim, 2012) Peanut

kernel contains 47-53% oil and 25-36% protein (Prasad et al., 2010)

These waste materials have the small valuable economy and often see as

a disposal problem As a result, the method improvement is really needed to manage the huge amount of peanut shell, which lead to a serious problem in waste management systems and afterward poses environmental pollution

(Westendorf, 2008) Changing peanut (Arachis hypogeal L.) shell into a

valuable raw material, ingredient or product would be the better method to make

use of them (Zhao, Chen & Du, 2011) As mention by Wilson et al., (2006),

peanut shell is low in density and high in volume and are used in animal feed or burned for energy Peanut shell has been stated to be promising effective material for the removal of heavy metal from wastewater (Imamoglu & Tekir,

2008; Oliviera et al., 2009; Brown et al., 2000) The chemical composition of

peanut shell, it is listed in Table 5

Table 5 Chemical composition of peanut shell Chemical Composition Percentage (%)

(Source: Kerr et al., 2006)

` The high cellulose content in peanut shell is biomass which it has a large adsorption capacity in the absorption of cationic compounds but it has small adsorption for anionic compounds because the biomass surface has a negative site Cellulose contains an active site that is a hydroxyl group which can form a series

of chemical reactions and bind with cationic and anionic compounds (O'Connell, Birkinshaw & O’Dywer, 2008) The hydroxyl groups in cellulose have the ability

to create hydrogen bonds between chains that function in the linear configuration

of cellulose chains Cellulose can bind with phosphate to form derivative phosphate which is selective for the OH position at C6 with a substitution degree

of about 0.2 (Heinze, El Seoud & Koschella, 2018)

2.3 Histopathological Effects

The effects in lower-level biochemical and higher-level population are an intermediate biological organizational level (Adams, Greeley & Ryon, 2000) Reproductive changes (in growth or reproductive parameters) usually take place slower than histopathological effects which give a better health assessment than a

single biochemical response (Segner & Braunbeck, 1988; Triebskorn et al., 1997)

Atabati et al., (2015) conducted research about effects of copper-(II)-sulfate

on gill histopathology of grass carp (Ctenopharyngodon idella) In this experiment,

the fish were exposed to 2.5 mg/L and 5 mg/L solutions of CuSO4 for 96 hours and during the experimental period, fish had not been fed On 2.5 mg/L solutions of CuSO4, there were lifting of the lamellar epithelium, edema in the filamentary epithelium and Red Blood Cell (RBC) exudation (RBC) RBC occurred due to lifting and necrosis of lamellar epithelium Moreover, the gills of grass carp in this concentration also, presented lamellar fusion in many areas because of filamentary epithelium proliferation The fish samples exhibited the biggest epithelial lesion signs of epithelial lesions on 5 mg/L solutions of CuSO4 Thereto, few aneurysms

were observed at gill lamellae The filament and lamellar epithelium were usually affected by necrotic cells and macrophages

Gills are acknowledged as one of critical organs to fish due to the primary place for respiration, ion regulation, and metabolic waste products discharge Also gills are well-known as gills are also known to be the first pollutant target because

it has a large area which is open to the external environment (Rankin & Jensen, 1993; Olsson & Kille, 1998) Due to high concentration of copper, it was reported

to cause the severe histopathological alteration in gills of teleost fish

(Figueiredo-Fernandes et al., 2007) According to Lease et al., (2003), monitoring fish health

in the milieu with gill histopathology is very useful as an early indicator The histopathological changes, that cause by waterborne toxicants, could be characterized quantitatively through morphometric methods (Cerqueira & Fernandes, 2002)

According to Alkobaby & Wahed (2017) research about the acute toxicity

of copper to Nila Tilapia (Oreochromis niloticus) fingerling and its effects on gill

and liver histology, all fish were exposed to 0 (control), 5, 10, 15, 20, 25, 30, 35 and 40 mg/L of copper-(II)-sulfate with 3 replications per treatment Fish, which exposed to the different concentration of CuSO4, showed epithelial lesions, epithelial hyperplasia of both primary and secondary lamellae epithelium, lifting

of the lamellar epithelium, edema in the filament of epithelium, curling and clubbed tips of secondary lamellae The severity of hyperplasia increased with the increase of copper-(II)-sulfate concentration, causing the full merger of several secondary lamellae

For appraising, the copper toxicity impacts have been exerted on histopathological and morphometric rat test modifications in Babaei, Kheirandish

& Ebrahimi (2012), which it was the experiments with 45 Wistar albino male adult rats (200-240g) The rats were randomly allocated to either control or two treatment groups each containing 15 rats The first treatment group was given copper-(II)-sulfate at a dose of 100 mg/kg in 0.2 cc and the second treatment group received copper-(II)-sulfate at a dose of 200 mg/kg in 0.2 cc As a result, the most

of the seminiferous tubules were degenerated and a few of them seemed to be relatively normal on the 28th day but on the 56th day, nearly all the seminiferous tubules were completely degenerated as only a single layer of certoli cells and spermatogonia was present

Species : Oreochromis niloticus

Binomial name : Oreochromis niloticus (Linnaeus, 1758)

The Nile Tilapia (O niloticus) has a profoundly body fish of cycloid scales

and silver in color with olive/grey/black body bars when the breeding season

happens, the Nile Tilapia flushes red frequently (Picker & Griffiths, 2011) The maximum length of Nile Tilapia is 62 cm with weighing of 3.65 kg which has at

an estimated 9 years of life (FAO, 2012) For O niloticus, the average size/total

length is 20 cm (Bwanika et al., 2004)

Central Africa, North Africa, and the Middle East are the native places

where O niloticus is originated (Boyd, 2003) Nile Tilapia is a rainy tropical

freshwater and estuary species which prefers shallow still waters on the lakeside and wide, sufficiently vegetated rivers (Picker & Griffiths, 2011) Fishery and Aquaculture Country Profiles of Egypt (2010) reported that after carps, the second most important group of farmed fish and the most widely grown of any farmed fish is Tilapias (including all species) in worldwide further, Tilapias (including all species) is the world's most widely cultivated of all farmed fish, and Egypt is the second largest farmed Nile Tilapia producer considered to be the commonest fish currently being, cultured commercially By far the most important species is Nile

Tilapia or O niloticus (El-Sayed, El-Ghobashy & El-Mezayen, 2013)

CHAPTER III METHODS

3.1 Place and Time

This study has been carried out from May 2017 to September 2017 The bioadsorbent adsorption process and toxicity test was conducted at Laboratory of Aquaculture, Faculty of Agriculture, Sriwijaya University, Indralaya Campus Histopathology examination was conducted at the Laboratory of Anatomical Pathology Laboratorium Barokah Palembang Meanwhile, adsorbent adsorption examination was conducted at Fish Production Technology Laboratory, Faculty of Agriculture, Sriwijaya University, Inderalaya Campus

3.2 Materials & Equipment

3.2.1 Toxicity Testing

The test species used in this toxicity test were 30 fish of Nile Tilapia

(Oreochromis niloticus) with weight of ± 30 grams and length of 6-8 cm Other

materials were CuSO4 pro analyst The equipment used; (a) an aquarium measuring 30 × 30 × 30 cm with an aerator to distribute oxygen to fish, (b) glass beaker, Erlenmeyer, gloves, mask, spatula, and tweezers for making stock solution

of Cu2+ and (c) fish strainer to take the dead fish from the aquarium

3.2.2 Adsorbent Testing

The materials were 300 fish of Nile Tilapia (Oreochromis niloticus) with

the weight of ± 30 grams and the length of 6-8 cm, distilled water, peanut shell, grade 42 paper filters, and stock solution of Cu2+ While the equipment used; (1) Atomic Absorption Spectroscopy (AAS), to see the remaining Cu2 + levels, (2)

analytical balance, to weigh the adsorbent and CuSO4 pro analyst that will be used, (3) aquarium measuring 30 × 30 × 30 cm with aerator, (4) glass breakers, Erlenmeyer, gloves, pipettes, masks, and magnetic funnels that used in the Adsorbent Experiment Using Fish as Bioindicator and Adsorbent Adsorption Examination, and (5) shakers which used to mix and stir Cu2 + solutions with adsorbent.

3.2.3 Histopathology Examination

In histopathology examination, the materials were (a) the specimens of gills

organ from Nile Tilapia (Oreochromis niloticus), (b) alcohol 70%, 95%, and 100%

to use in the washing process, (c) Hematoxylin Eosin used for coloring, (d) Bouin's

as a fixative solution, (e) paraffin was used for impregnating and embedding, (f) tissue, for cleaning, and (g) xylene, used for clearing the slide The equipments used in histopathology examination, namely:

a Glass objects, to place preparations

b Scissors, for cutting organs

c Scalpel, to dissect organs

d Tweezers, for taking organ samples

e Petri dish, to put the preparations

f Sample bottle, to preserve the tissues

g Cassette and deckle, to block paraffin containing samples

h Microtome, to cut the tissues

i Trays, to dissect samples

j Microscope, to see the damage on organ tissue

3.3 Preparation

3.3.1 Fish Preparation

Oreochromis niloticus or Nile Tilapia was obtained from Fish Hatchery

Center Indralaya, Ogan Ilir Fish were selected based on the size (6-8 cm) The size of fish was equalized in order to avert significant difference in the results of the treatment and also replication in this study The fish were acclimated in laboratory conditions for 7 days prior to adapt and the selected fish for the experiment were the fish that swam actively, healthy, and in good condition

3.3.2 Preparation of Cu 2+ Solution

Stock solution of Cu2+ ion was prepared by weighing 2.5g of CuSO4 pro analyst 1000 mL of distilled water was added into beaker glass after that, it was stirred well by magnetic stirrer Using diluted formula, the stock solution was used

to make a fixed amount of Cu solution with concentration 20 mg/L, 25 mg/L, 30 mg/L, and 35 mg/L that used for preliminary toxicity test Diluted formula:

Vstock x Cstock = Vcalibration x Ccalibration

Where:

Vstock = Volume of Cu2+ stock solution

Cstock = Concentration of Cu2+ stock solution

Vcalibration = Desired volume of Cu2+ solution

Ccalibration = Desired concentration of Cu2+ solution

3.3.3 Adsorbent Preparation

The peanut shell was initially washed with water and separated from other dirt Afterwards, the cleaned and dried peanut shell were burned at 250°C for 2.5 hours After cooling, the peanut shell ash was slightly sieved by 212 μm mesh after drying Finally, the ash from sieving was stored and this peanut shell ash was used for the adsorbent in the heavy metal adsorption process (Nurhasni, Hendrawati & Saniyyah, 2014)

3.4 Methods

3.4.1 Toxicity Testing

Preliminary toxicity test was run to decide LC50 of copper-(II)-sulfate (CuSO4) in Nile Tilapia When conducting toxicity testing, there were 5 aquariums that contained 10L of water and 10 fish 4 aquariums were exposed to the copper-(II)-sulfate (CuSO4) and for the control, one aquarium was not exposed to the chemical The concentrations used for toxicity testing were 20 mg/L, 25 mg/L, 30 mg/L, and 35 mg/L which were considered as test solutions During toxicity test, fish were not fed LC50 value is measured by using probit analysis (SPSS)

3.4.2 Adsorbent Experiment Using Fish as Bioindicator

This adsorbent experiment was aimed for the potentials of peanut shell ash

as adsorbent on removal of heavy metals (Cu2+) in the water The experimental procedures were conducted in the glass aquaria which consisted of 10 L water and the mixture of Cu2+ ion After acquiring the concentration that caused the death of 50% of the fish population (LC50), the concentration was varied to: concentration

of LC50 minus 20% of LC50 (C-20%); LC50 concentration minus 10% of LC50

(C-10%); concentration of LC50 (C); concentration of LC50 plus 10% of LC50 (C + 10%); and LC50 concentration plus 20% of LC50 (C + 20%) 40 grams of peanut shell ash were inserted for each glass aquaria that contained 10 L water with various CuSO4 concentrations as mentioned earlier Peanut shell ash, Cu2+ ion, and water were well mixed together and left for 24 hours to separate the residue and the water (Dade Jubaedah, 2017)*

After 24 hours, the residue inside the glass aquaria was removed and then

10 fish, that were chosen randomly, were inserted into each aquarium The dead fish were carefully taken out from aquarium to avoid contamination that could affect other fish Afterward, the portions of gills were removed for histopathological examination

3.4.3 Adsorbent Adsorption Examination

For doing Adsorbent Adsorption Examination or AAS, the aim was to investigate the adsorption of heavy metal (Cu) onto peanut shell ash 1 gram of peanut ash were weighed into 250 mL Erlenmeyer Then, 100 mL of the solution contains 2.2 mL of ion CuSO4 were mixed to the adsorbent Erlenmeyer was covered up with aluminum foil and was shaken using shaker 200 rpm for 30, 60,

90, 120, and 150 minutes Afterwards, between the adsorbent mixture and the solution was separated using whatman 42 filter paper The final filtrate product from Cu solution adsorbent was evaluated with AAS (Atomic Absorption

Spectroscopy) (Qaiser, Saleemi & Ahmad, 2007; Prasad et al., 2008)

3.5 Histopathological Examination (Raphael, 1976; Bancroft & Gamble, 2002)

Histopathological examination was conducted by technicians in the Laboratory of Anatomical Pathology of Laboratorium Barokah Palembang The required procedures of histopathological examination include these following steps:

of tissue processing

3.5.3 Sectioning

In sectioning processes, sections were cut 5µm thickness by microtome The ribbon of section was placed onto a warm water floating bath where the bath temperature was ~45ºC in order to remove the wrinkles without melting the ribbon Then The water sections were mounted on a glass microscopic slide After that, the 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 Next, the slides

were ready to continue for staining process

3.5.4 Staining procedure using Hematoxylin and Eosin

Due to staining process cannot be done if it still contains paraffin wax Then

to dissolve the paraffin wax, the slides were immersed in xylene, a hydrocarbon

solvent by passing through various alcohol 95%, 90 %, 80 %, 70 %, 50 % and 30

% for 5 minutes each one after the other The next step, the slides were washed off

in running tap water for 5 minutes and stained using hematoxylin for 10 minutes

Afterward the slides were rinsed in running tap water for 10 minutes Then, they

were stained by Eosin from 15 seconds to 5 minutes Following this, the stained

slides were dehydrated once more time by passing them through an ascending

series of alcohol 30%, 50 %, 70 %, 80 %, 90 % and 95 % for each 3 minutes For

the next step, the slides were dipped from 2 to 3 times in 95% of alcohol and then,

in 100% alcohol for 1 to 2 minutes each After that, the slides were placed in

acetone and two changes were provided for 3 minutes each Next, the stained slides

were dipped in xylene (absolute alcohol with ratio of 1:1) and two changes were

carried out for each 3 minutes The slides after clearing with xylene were mounted

in Dibutyl Phthalate Xylene (DPX) medium The stained slides were examined

under microscope and photographed at both high as well as low power resolutions

The nuclei stained blue and cytoplasm in different shades of pink Finally, the

stained slides were investigated for histopathological alterations

CHAPTER IV RESULTS AND DISCUSSION

4.1 Preliminary Toxicity Test

This toxicity preliminary test was aimed to obtain a concentration range that caused the mortality of 50% population of animal testing, or what is known as Lethal Concentration (LC50) The preliminary test was carried out on Nile Tilapia with 10 animals testing for every concentration and the length of fish was around 6-8 cm There were 5 aquariums containing 10L of water and 10 fish which were randomly selected for each aquarium Four aquariums were exposed to copper (II)

- sulfate (CuSO4) and one aquarium was not exposed to chemicals and it used as control The concentrations used for toxicity testing were control (0 mg/L), 20 mg/L, 25 mg/L, 30 mg/L and 35 mg/L From preliminary toxicity test, the collected data are listed at the Table 6:

Table 6 Preliminary Toxicity Test Result

From the preliminary test of toxicity to fish mortality was obtained the data (Table 6) In the data table, it could be seen that the highest number of fish deaths were at the highest concentration of 35 mg/L with a mortality rate of 100% At concentration of 25 mg/L and 30 mg/L, the fish died as many as 8 fish and 9 fish consecutively while at concentration of 20 mg/L the death fish was found as 4 fish However, the death of fish was also found in the aquarium with control treatment,

Concentration Population Mortality Mortality Rate (%)

which there was no chemical, as many as 1 fish that could be due to stress factors

on the new environment During this preliminary test, the needed time for almost all fish to die was around 4 to 6.5 hours followed by behavioral changes in Nile Tilapia

The presented data in table 6 shows that fish mortality is occurred due to the exposure of CuSO4 contained in water where it affects organ system in fish, it is clearly evident that copper-(II)-sulfate has a role in fish mortality This proves that the more concentration given, the higher fish mortality rate occurs because of the hazardous content of CuSO4 Fish death is occurred due to the damage of mitochondria where it is one of the cell organelles and has a function as the place where respiration takes place on living things and also uses to produce energy Exposure of Cu causes an increase in the release of metabolism which has the function of detoxification and also maintains homeostasis which refers to the state

of steady or dynamic balance in the organism's body According to Kim and Kang (2004), the body had a check and balance function thereby when the amount of Cu was exceeded inside the body, the production of proteins that bind to metals such

as metallothionein and ceruloplasmin would also be increased This protein production depleted the body's energy needs and the effect is the reduction of energy used for somatic growth In addition to the reduced in energy production, the hemoglobin level also decreases and results in the fish capability to provide oxygen that is needed by body tissues to become increasingly limited until fish activity gradually decreases and then die This demonstrates that Cu which is

supposed to play a role in hemoglobin synthesis, but on the contrary it actually inhibits hemoglobin synthesis

To obtain the probit value of LC50, the data from Table 6 was processed using the SPSS program so that the obtained data information for LC50 was 21.91 mg/L (Appendix 1) The value of concentrations that has been varied is shown in Table 7:

Table 7 The Value of Varied Concentrations

4.2 Adsorbent experiment using fish as bioindicator

This adsorbent experiment was conducted to determine the effectiveness of peanut shell as a natural adsorbent to absorb the heavy metals Peanut shell that had been processed into adsorbents through the physical activation were put into

an aquarium containing 10L of water containing CuSO4 with the specified concentration in Table 7 for 24 hours in order to make the pollutant absorption process more effective After 24 hours, the adsorbent turned out to settle at the bottom of the aquarium and the water color in the aquarium becomes much clearer when compared to treatments that did not use adsorbents (Appendix 14,15) where the previous water color is more bluish The change in water color had become a positive effect of using peanut shell as natural adsorbent to reduce Cu levels despite its important role in cell metabolism because Cu is very toxic to aquatic animals if

Varied Concentration

After filtering the adsorbent which was at the base of the aquarium, the next process was continued, it was using fish as bioindicator to determine whether heavy metal adsorption of Cu using peanut shell as adsorbent was effective or not compared to treatment without adsorbent The percent mortality of fish in different concentrations of copper-(II)-sulfate was determined at 48h exposure For this, the experimental fish were divided into batches of ten each, and were exposed to different concentrations of copper-(II)-sulfate ranging from 17.53 mg/L to 26.29 mg/L and mortality rate was observed and recorded for all the concentration after

48 hours A batch of fish was also maintained simultaneously in treatment without adsorbent, which served as negative control All the experiments were repeated five times to confirm the results Fish mortality for each replication can be seen in Table 8

Table 8 Effect of CuSO4 (Copper-(II)-sulfate) on Nile Tilapia in Five