Chapter 1

THE PROBLEM AND ITS BACKGROUND

Introduction

Water pollution affects plants and organisms living in these bodies of water and, in

almost all cases the effect is damaging not only to individual species and populations, but also

to the natural biological communities. Water pollution occurs when pollutants are discharged

directly or indirectly into water bodies without adequate treatment to remove harmful

compounds. Direct sources include effluent outfalls from factories, refineries, waste treatment

plants etc.. that emit fluids of varying quality directly into urban water supplies. On the other

hand, indirect sources include contaminants that enter the water supply from soils/groundwater

systems and from the atmosphere via rain water. Soils and groundwaters contain the residue of

human agricultural practices (fertilizers, pesticides, etc..) and improperly disposed of industrial

wastes. Atmospheric contaminants are also derived from human practices (such as gaseous

emissions from automobiles, factories and even bakeries).

In the Philippines, the continuing technological industrialization is a booming business in

most urban areas. The production of local materials for all aspects of products: medical, leisure,

kitchenware, tools, etc. The products are composed of toxic chemical and metals. Heavy and

toxic metals are most important because of their non-biodegradability. Industrial wastes are the

major sources of copper in the environment. Copper, ranking 25th in abundance is a minor

element of the earth’s crust (Wedepohl, 1995). Copper is one of the inorganic contaminants that

the Environmental Protection Agency regulates in ground water and drinking water. It is listed

under the National Primary Drinking Water Regulations for inorganic chemicals along with

common contaminants such as chromium, lead, mercury, and cadmium. (EPA, 2006). Copper is

essential to human health, however, to much exposure to the element can be dangerous

(Huang, 2007). Excess intake of copper would cause acute and chronic adverse health effects

such as stomach and intestinal distress, liver and kidney damage, and anemia (EPA, 2006).

Conventional copper removal methods are ion exchange, chemical pecipitation, ultra

filtration, and electrochemical deposition (Aslam et al., 2004). These copper reduction methods

are expensive and are not environmental friendly because these require additional chemicals,

and therefore, would increase the volume of chemical and biological sludge. (Kratochvil and

Volesky, 1998). Furthermore, these technologies work best at wastewater with very high

concentration of heavy metals (Esmaeili, 2009). Compared with conventional methods for

copper reduction, biosorption is a better alternative. Some of the biomass that are proven to

sequester heavy metals are Aspergillus niger, Aspergillus terreus, Rhizopus oryzae, Penicillium

chrysogenum, Metarrhizium anisopliae var. anisopliae and Penicillium verrucosum.

Biosorption is the process that makes use of living or dead biomass to absorb heavy

metals from aqueous solutions. However, use of living biomass is difficult for it requires a

continuous supply of nutrients.

Background of the Study

Pollution is not a new crisis to the changing society. The factories that emit thick clouds

of smoke and chemical-induced liquids affect the communities near the area. Not only are

plants’ growth affected, but also animal reproduction and health. The humans suffer too. They

have to inhale and intake these toxic materials. Most people living near the sewers are those

with low standards of living, and illegal settlers. They are economically deprived and they have

no fortune for medical deficiencies. Overdoing of disposal of chemicals and heavy metals from

factories in industrial wastewaters can cause water contamination. This concerns the people

Milkfish (Chanos Chanos) Scales

Mass of Biosorbent:4 grams8 grams

Concentration of Copper (mg/L)

living near those sewage wastewaters. Since heavy metals like copper are harmful to human

health and can be associated with diseases in liver and kidney, gastrointestinal damage and

mental retardation in children. It is now time to research methods that would sequester these

metals before it will affect the health of several people. Many different processes and

technologies are being used to lessen the contamination of these harmful substances in the

wastewaters. Biosorption a new technology that is being used today would likely be the most

suitable process that can sequester the metals since it uses the ability of biological materials.

The proponent prefer this method because of its effectiveness in reducing the concentration of

heavy metal ions to very low levels, the use of inexpensive biosorbent materials, minimization of

chemical and or biological sludge, no additional nutrient requirement, regeneration of

biosorption and possibility of metal recovery. It is also an effective alternative to physic-chemical

methods of separation in treating wastewater contaminated with heavy metal ions. Extensive

efforts have been made to explore new types of biosorbent materials.

A recent study done by Huang (2007) suggested that fish scales of Tilapia nilotica

Linnaues, can be a better alternative to reduce the level of copper. Following Huang (2007), this

study will investigate the sorption capabilities of fish scales of Chanos chanos for the uptake of

copper in water. The advantages of using fish scale as a biosorbent are cost-effective, minimal

energy usage and is environmentally-friendly. With these advantages, wastewater treatment

facilities could adopt the use of fish scales in biosorption processes. Moreover it will lessen the

risk of diseases among people living near industrial plants.

Conceptual Framework

Independent Variable Dependent Variable

In Fig 1.1 the research paradigm shows that the independent variable, the mass of the

biosorbent which is the milkfish scales, will affect the concentration of copper which is the

dependent variable under the study.

Statement of the Problem

The objective of this study is to investigate the copper sorption capabilities of fish scales

of Milkfish (chanos chanos) with ratios of the amounts of fish scales to the initial concentration

of copper. Specifically, the study includes the following goals:

1. Is there any significant difference in the concentration of copper with and without the

addition of biosorbent in the copper solutions?

2. Is there a significant difference in the concentration of copper by adding the following

amounts of biosorbent in the copper solutions?

a. 4 g

b. 8 g

Hypothesis

1. There is no significant difference in the concentration of copper with and without the

addition of biosorbent in the copper solutions?

2. There is no significant difference in the concentration of copper by adding the following

amounts of biosorbent in the copper solutions?

a. 4 g

b. 8 g

Significance of the Study

This study aims to lessen water contamination of toxic waste products from industries. It

is viewed that the investigation will contribute to new information and utilization of biosorption in

eradicating copper from toxic releases and to explore a biosorbent material that has the

capability to attract and sequester the metals. Also, this study may help in our environment

specifically in industrial wastewater by lessening high concentration of copper that contributes to

water contamination. The process biosorption used in the study is also eco-friendly since the

study would make use of biological materials that are not harmful to the environment.

Advantages of using fish scale as a biosorbent are cost-effective, minimal energy usage and is

environmentally-friendly. With these advantages, wastewater treatment facilities could adopt the

use of fish scales in biosorption processes.

Moreover, this study lies on the purpose of helping people in the community have a safer

means of purifying wastewater, and to contribute in lessening the problem of water pollution in

our environment by using fish scales which are already wastes generated from households and

markets.

Scope and Limitations of the Study

This study will be conducted for at least one week, including the testing of the solutions

in the Ultraviolet-visible Spectroscopy in Adamson University and Electron Microscopy for the

comparing and analysis of the results.

The variables of the study specifically the copper solutions and milkfish scales will be

obtained from Bambang and Munoz wet market. The materials that will be needed is copper

solution which was prepared with an initial concentration of 5000mg/L. Glacial acetic acid will be

used for the buffer and 0.1M NaOH will be used to adjust the buffer to the desired pH=4 ± 0.5.T

The instruments will be used in the study were for filtration of the solutions and to determine the

amount of absorbed copper. The sorption experiments will be conducted in six 125mL

Erlenmeyer flasks containing 100mL of the copper sulfate solution. And for the filtration process

a Büchner funnel, filter flask and a clamp. The determination of the amount of copper amount in

the solutions will be the use of the Ultraviolet-visible Spectroscopy and Electron Microscopy.

Definition of Terms

To provide a vivid frame of the reference and better understanding to this study, the

following terms are defined based on how each term were used in the study.

Biosorbent – fish scales of milkfish (Chanos chanos) used to remove copper from the copper

(II) sulfate solution.

Biosorption – a process that binds concentrated heavy metals (copper) by using a biosorbent

material (milkfish scales).

Copper – the heavy metal observed in the study.

Electron Microscope- an instrument used to determine the relative amount of the copper in the

fish scales.

Spectrophotometer – an analytical laboratory assessment procedure used to determine the

initial and final concentration of copper in the copper (II) sulfate solution.

Foreign Literature

Water Pollution and Heavy Metal pollution

Water pollution affects plants and organisms living in these bodies of water and, in

almost all cases the effect is damaging not only to individual species and populations, but also

to the natural biological communities. Water pollution occurs when pollutants are discharged

directly or indirectly into water bodies without adequate treatment to remove harmful

compounds.

This is a major environmental issue affecting many nations. There are three types of

pollutants: physical, chemical, and biological. Physical pollutants include sediments, sewage

plant sludge, opacity and radiation. Biological pollutants include human excrements, plants,

bacteria, fungi and parasites. Chemical pollutants abound in ambient and drinking water; they

include petroleum products, dissolved solids, phosphate, chloride, nitrate fluoride, metals,

organic material, asbestos, and other carcinogens.

Generally speaking, pollution in the environment has concerned itself of how to stop and

diminish the pollutants. Due to industrialization different pollutants particularly heavy and toxic

metals that are being released into the environment has increased particularly in the industrial

wastewaters. Using tools and methods such as controlled laboratory experiments and the new

technology involving the removal of toxic metals from the wastewaters which is known as

biosorption. Biosorption can be one solution to clean up heavy metal contamination.

Researchers have considered the advantages of using this technology in sequestering the

heavy metals.

Main sources of heavy metal contamination include urban industrial aerosols, solid wastes

from animals, mining activities, industrial and agricultural chemicals. Heavy metals also enter

the water supply from industrial and consumer water or even from acid rain which breaks down

soils and rocks, releasing heavy metals into streams, lakes and ground water.

It has been established that dissolved metals (particularly heavy metals) escaping into

the environment pose a serious health hazard. They accumulate in living tissues throughout the

food chain, which has humans at its top, multiplying the danger. Overt signs of acute

intoxication include dullness, restlessness, irritability, poor attention span, headaches, muscle

tremor, hallucinations, and loss of memory, with encephalopathy occurring at blood lead levels

of 100–120 μg dL-1 in adults and 80–100 μg dL-1 in children.

The toxicity of metal ion is because of their ability to bind with protein molecules and

prevent replication of DNA and therefore, demote cell division (Kar et al., 1992).

Imbalances or excessive amounts of a metal species along this lead to toxicity

symptoms, disorders in the cellular functions, long-term debilitating disabilities in humans, and

eventually death. One of the most insidious effects of inorganic lead is its ability to replace

calcium in bones and remain there to form a semi permanent reservoir for long-term release

well after the initial absorption. Such on-site treatment of waste streams contributing a load of

heavy metals would be more efficient than treating the large volumes of mixed, diluted metal-

contaminated waste water in a general (municipal) sewage treatment plant (Naja et al., 2003)

Copper

Copper, one of the most widely used heavy metals, is mainly employed in electrical and

electroplating industries and in larger amounts and is extremely toxic to living organisms. The

presence of copper (II) ions, cause serious toxicological concerns, it is usually known to deposit

in brain, skin, liver, pancreas and myocardium (Davis et al., 2000).

Based on the study of Huang (2007), copper is one of the inorganic contaminants that the

Environmental Protection Agencyregulates in ground water and drinking water. It is listed under

the National Primary Drinking Water Regulations for inorganic chemicals along with common

contaminants such as chromium, lead, mercury, and cadmium (EPA, 2006). Copper is naturally

deposited in rocks as mineral form, which is mostly associated with sulfur (USGS, 2007).

Anthropogenic sources of copper include the production of plastic material, copper and other

nonferrous smelting, and steel blast furnaces. The major source of copper contamination in

drinking water is from plumbing material, due to the corrosion of copper pipes from passing

water through these pipes (EPA, 2006). Copper released into the environment indefinitely

persist, circulate and accumulate in the food chain (Vijayaraghavan et al., 2004).

Biosorption

Biosorption is the process which makes use of living or dead biomass to sequester

heavy metals, even from very dilute solutions (Volesky, 1990). Moreover, biosorption does not

promote the chemical sludge, which is a major problem for removal of heavy metals.

Seaweeds, especially brown algae, have shown high metal uptakes (Davis et al., 2003).

In sequestering heavy metals, nitrate salts have been a good help. Nitrate in solutions are less

likely to form complexes with metals that can be present as free metal ions (Diniz et al., 2001).

Fourest and Roux’s(1992) definition of biosorption is the ability of biological materials to

accumulate heavy metals from industrial wastewater though metabolically mediated or

physicochemical pathways of uptake. Shumate and Stranberg (1985) on the other hand, define

it as a non- directed physicochemical interaction that may occur between metal/radionuclide

species and microbial cells.

Some researches on biosorption revealed that biosorptn is a occurrence wherein the

metallic species are deposited in the solid biosorbent through different sorption processes

(Ercole, 1994).

There are certain factors that affect biosorption. One of these is the temperature. Unless

the solution is in the range of 20-35°C, the biosorption performance will be influenced (Aksu et

al., 1992). pH is the most important factor in biosorption for it affects, not only the solution

chemistry of the metals, but the activity of the functional groups in the biomass and the

competition of metallic ions (Friis and Myers-Keith, 1986). Moreover, the concentration of the

biomass also affects the specific uptake (Fourest and Roux, 1992).

Moreover, biosorbents, especially the saturated ones, can be reused after. The

deposited minerals can be recovered. The metals are released in a concentrated wash solution

that will also regenerate the biosorbent (Volesky, 2007).

Biosorbent

There are certain chemical groups that can sequester and accumulate metals:

acetamido groups of chitin, structural polysaccharides of fungi, amino and phosphate groups in

nucleic acids, amino, amido, sulphydryl and carboxyl groups in protein, hydroxyl in

polysaccharide and mainly carboxyls and sulphates in polysaccharides of marine algae (Ahalya

et al., 2003). According to Bux and Kasan (1994), the higher the electronegativity of the

biomass, the greater the attraction of heavy metal cations.

Foreign Studies

A study done by Mustafiz et al. (2003) suggested that fish scales of Atlantic cod, Gadus

morhua Linnaeus, be a better alternative to reduce the level of lead, arsenic and chromium in

water. According to his study, the uptake abilities of scales from different fish species should be

similar because most fish scales contain significant portions of organic protein (collagen), and

the structure of collagen shows that it contains the possible functional groups, such as

phosphate, carboxyl, amine and amide, that are involved in the biosorption of heavy metals

(Mustafiz et al., 2003).

Following the study of Mustafiz (2003), Huang (2007) investigate the sorption

capabilities of fish scales of Tilapia nilotica Linnaues for the uptake of copper in water. Based on

his results, as the amount of biosorbent material added is increased, the sorption percentage

also increased. Also as the contact time is increased, the sorption percentage also increases.

As reported by Badel, et.al (2003), the removal of heavy metals (Cadmium, Zinc and

Lead) from aqueous solution was possible using a keratin powder prepared from Algerian sheep

hooves. It was seen that adsorption took place for the three metals on 90 minutes for Cd and 60

minutes for Zn and Pb. Under our experimental conditions; pH plays an important role in the

adsorption process, particularly on the adsorption capacity. The pH of the solution has a great

influence on the adsorption capacity of keratin for the studied heavy metals. The pH selected to

give an optimum rate of adsorption are 8.5, 7 and 5.6 respectively for Cd, Zn and Pb. It is shown

that steamed hoof powder issued from Algerian sheep hoofs has a relatively high adsorption

capacity for these heavy metals; the quantities adsorbed per gram of steamed Algerian sheep

hoof powder at equilibrium (qe) are 29.5 mg / g for Cd and 42.5 mg / g for Zn and 65 mg / g for

Pb. This adsorption is described by an isotherm of type I and is fully verified by the Freundlich

isotherm. The kinetics of the Cadmium, Zinc and lead adsorption on the steamed Algerian

sheep hooves was found to follow a pseudo-second-order rate equation.

The larger the surfaced area, the more Cu was physically adsorbed onto the biosorbent.

However, if Cu is removed physically from water, further treatment should be applied to extract

or retrieve the Cu from the used fish scales. Directly disposing the used fish scales as raw

garbage defeats the purpose of the biosorption process, because the Cu could be re-released

into the environment.

The other possible role contributing to Cu removal was microbes in the fish scales. The

study done by Mustafiz et al. (2003) suggested that microbes were responsible in heavy metal

removal with the application of fish scales as a biosorbent. It was possible that a longer contact

time allowed the microbes to be released into the Cu solution. The microbes needed to be in

solution for a longer time in order to absorb Cu. As the result, shorter contact time did not allow

enough time to trigger the Cu sorption abilities of the microbes in the biosorbent.

II.Biosorbent

Biomass can come from: fast-growing organisms that are specifically cultivated or

propagated for biosorption purposes (crab shells, seaweeds). (Ahalya, Ramachandra and

Kanamadi 2003).Biomass exhibits a property, acting just as a chemical substance, as an ion

exchanger of biological origin (Yun, Park and Volesky 2001). The quality of the sorbent material

is judged according to how much sorbate it can attract and retain in an ‘immobilized’ form. For

this purpose it is customary to determine the metal uptake by the biosorbent as the amount of

sorbate bound by the unit of solid phase (by weight, volume, etc.). Correspondingly, the amount

of metal bound by the sorbent which ‘disappeared’ from the solution can be calculated based on

the mass balance for the sorbent in the system

Biosorbents can accumulate in excess of 25% of their dry weight in deposited heavy

metals: Pb, Cd, U, Cu, Zn, even Cr and others. Due to higher affinity of the sorbent for the

sorbate species, the latter is attracted and bound there by different mechanisms. The process

continues till equilibrium is established between the amount of solid-bound sorbate species and

its portion remaining in the solution. (Volesky 2000).

While there is a preponderance of solute (sorbate) molecules in the solution, there are

none in the sorbent particle to start with. This imbalance between the two environments creates

a driving force for the solute species. The heavy metals adsorb on the surface of biomass thus,

the biosorbent becomes enriched with metal ions in the sorbate. The degree of sorbent affinity

for the sorbate determines its distribution between the solid and liquid phases.

The importance of any given group for biosorption of a certain metal by a certain

biomass depends on such factors as the number of sites in the biosorbent material, the

accessibility of the sites, the chemical state of the sites (i.e., availability), and the affinity

between the site and the metal (i.e., binding strength). Both chemical pretreatments, such as

contacting cells with acids, alkali, and organic compounds and physical pretreatments, such as

heat treatment, autoclaving, freeze drying, and boiling and showed enhancement in metal

biosorption by microorganisms.

Local Literature

Successive treatment of loaded biomass can enable recovery of valuable element or

further containment of highly toxic species. Heavy metals from liquid wastes and results in safe

environmental discharge (Mamaril, Foser and Alphante 1997). Biosorbents are one material that

binds with metals (Mojica and Palafox 2004).

Local Studies

Biosorption is at industrial interest because of the removal of toxic heavy metals from

liquid wastes. Chitin is a polymer of N-acetyl B.D. glucosamine. Presence of the amine and

hydroxyl groups in chitin serve as binding sites for lead ions. (Mojica and Palafox 2004).

Among the possible advantages of biosorption that is being low cost over other

technologies such as an ion exchange and reverse osmosis ,its high efficiency to accumulate

and sequester the metal ions, minimization of chemical and or biological sludge, no additional

nutrient requirement since it make use of biological materials as adsorbents, regeneration of

biosorbent and possibility of metal recovery of the deposited metals from the saturated

biosorbent because they can often be easily released from the biosorbent in a concentrated

wash solution which also regenerates the biosorbent in a concentrated wash solution which also

regenerates the biosorbent for subsequent multiple reuse.(Kratochvil and Volesky 1998). This

makes the process highly economical and competitive particularly for environmental

applications in detoxifying effluents of metal-plating and metal-finishing operations, mining and

ore processing operations, battery and accumulator manufacturing operations, thermal and

nuclear power regeneration. Therefore, new types of biomass have been searched and

modified physically, chemically and even biologically to improve the metal sequestering

performance.

CHAPTER 3

Research Methodology

This chapter presents the procedures that will be employed the research design, product

formation, treatments, sampling, instrumentation, procedure in gathering data and statistical

tool.

Research Design

The researcher used the control group design to determine the biosorption activity of

milkfish (Chanos chanos) scales in removing copper in water. The study used control and

experimental groups in Phase III of the experimentation process.

Preparation of Biosorbent and Copper Solution

Scales of Milkfish (Chanos chanos), used as biosorbent in this study, were collected

from Munoz market. The biosorbent were soaked in distilled water for 24 hours and rinsed three

times with distilled water. The biosorbent was transferred to a container, air dried for two days

and stored at room temperature. All copper solutions used in this study were prepared by

adding 12. 57 g of copper(II) sulfate anhydrous in one liter of distilled water. The entire five liters

Cu stock solution was distributed in 9 different Erlenmeyer flasks, 100mL of Cu stock solution in

each flask, and stored at room temperature. Glacial acetic acid was diluted to make a 0.5M

acetate buffer with the pH=3.9, and 0.1M NaOH was used to adjust the buffer to the desired

pH=4 ± 0.5. The acetate buffer was used to maintain the acidity of the Cu solutions at the pH=4

± 0.5. Two milliliters of the acetate buffer was added to each sample before the biosorbent was

added.

Varying Biosorbent to Copper Ratios

All samples were treated at room temperature, and with pH=4 ± 0.5. The initial and final

copper concentrations ([Cu]), in units of milligrams of Cu per liter of solution, were determined

by Spectrophotometer.

The initial [Cu] of each sample was determined. Different amounts of biosorbent (4.0g,

and 8.0g) were added to six separate flasks containing Cu stock solution with constant initial

[Cu]. Different amounts of biosorbent represent different biosorbent to Cu ratio (BS:Cu) (Table

1). BS:Cu was calculated using the equation,

BS: Cu = mass of biosorbent = mass of biosorbent mass of copper initial [Cu] x volume of sample

The samples were shaken for two hours and was stored at room temperature for 24

hours. After the treatment, the biosorbent was filtered out using filter paper. Although no

biosorbent was added to the untreated sample, it was filtered before the determination of the

final [Cu] to control for any copper sorption caused by the filtering processes. The filtrate was

collected, then the final [Cu] was determined. The calculated initial and final [Cu] were used to

determine the specific uptake (Qeq), in units of gram of Cu removed per gram of biosorbent

used, and the sorption percentage (%S). Qeq and %S were calculated using the equations,

Qeq = (initial [Cu] – final [Cu]) x volume of samplemass of biosorbent

% S = (initial [Cu] – final [Cu]) x 100Initial [Cu]

Statistical Tool

The study will use T-test as its statistical tool

PHASE II:Preparation of the Copper Solutions

PHASE I:Preparation of the Biosorbent

Collection of Fish scales

Soaking in distilled H2O for 24 hours and rinsing three times

Air drying for two days and storing at room temperature

PHASE III:Varying biosorbent to copper ratios

Determination of initial copper concentration using spectrophotometer microscopy

Addition of different amounts of biosorbent (4.0g, and 8.0g) to six separate flasks containing Cu stock solution with constant initial [Cu].

Shaking the samples for two hours and sotring for 24 hours. Filtering the biosorbent using filter paper.

Determination of final copper concentration using spectrophotometer

PHASE IV:Statistical test

Using a statistical tool to determine the significant difference in the concentration of copper with and without the addition of biosorbents in copper(II)sulfate solution and with varying amounts.

Adding 12.57 g of copper (II) sulfate anhydrate in 1L distilled H2O

Distribution of stock solution 100 mL stock solution in 9 different Erlenmeyer flasks at room temperature.

Addition of 2 mL acetate buffer to each of the Erlenmeyer flasks. Using a data logger to determine the pHn of the solution

Figure 1: Schematic diagram