CHAPTER I

INTRODUCTION

Background of the Study

The increase in demand for oil due to industrialization

and urbanization has led to vast increase in the oil

exploration activities. Unfortunately, huge amount of oil

coming from the sea and even larger volumes are being

shipped making the marine and freshwater environment very

vulnerable to accidental releases and pollution problems.

Most of the oil spill accidents are brought about by the

malfunctions of oil tankers where most leads to large

amounts of oil being released into the bodies of water. On

the other hand, small amounts and gradual releases of oil

are caused by leakages on oil pipelines. Recently, a bunker

oil depot pipeline has leaked thousands of liters of bunker

oil into the Pasig River threatening to cause pollution and

environmental damage (Abejero, 2012; Estuaria and Viado,

2013).

Bunker oil spill is an ongoing environmental problem

because it is extremely hazardous to organisms and cleaning

up is also difficult. The damage consequently caused health

and economic problems, particularly, affecting local

fisheries, wildlife, and tourism.

1
 Phytoremediation has been gaining popularity as a

potential technique to solve environmental contaminations in

recent years(Haskell, 2014).Aquatic macrophytes such as

water hyacinth (Eichhornia crasssipes), water lettuce

(Pistia stratiotes), duckweed (Lemna minor), Esthwaite

waterweed (Hydrilla verticillata) and different species of

Azolla are utilized to remove, absorb or degrade heavy

metals and other contaminants (Gupta P., Roy and

Mahindrakar, 2012; Gupta R. and Tiwari, 2011; Rahman and

Hasegawa, 2011). This technology can serve as an alternative

clean up method for contaminated sites. It can be a

substitute for mechanical and conventional remediation

technologies that are often expensive and labor intensive.

The use of plants for bioremediation technique as treatment

for the oil spill is much safer. It avoids additional harm

and damage to the ecosystem and community hence

acceptability would not be an issue (Haskell, 2014).

In this research, Azolla pinnata R. Br. (water fern)

will be used as plant remediator. This plant is considered

as invasive or unwanted species but it employs a great

potential in bioremediation. Compared to other aquatic

plants like Pistia stratiotes (water lettuce) and Lemna

minor (duckweed), Azolla pinnata R. Br. (water fern)

possesses more of the properties of an ideal plant for the

2
use in phytoremediation such as fast growth rate, nitrogen

fixing ability, high biomass production, moderately

extensive root system, easy to harvest and high tolerance to

a wide range of heavy metals (Sood, Uniyal, Prasanna and

Ahluwalia, 2012, http://www.ncbi.nlm.nih.gov/pmc/

articles /PMC3357840/). In addition, Azolla pinnata R. Br.

(water fern) is smaller than Pistia stratiotes (water

lettuce) and Lemna minor (duckweed) thus reducing its

tendency to clog on waterways. According to the study done

by Al-Baldawi, Abdullah, Suja, Anuar and Idris (2012), this

fast-growing aquatic plant has the ability to degrade

hydrocarbons in their growth medium, making it as a

potential plant that can be used in phytoremediation. This

research intends to utilize Azolla pinnata R. Br. (water

fern) to provide a complete rehabilitation process and

science based response and management for oil spill.

Statement of the Problem

This study will investigate the ability of Azolla

pinnata R. Br. (water fern) to reduce bunker oil

concentration.

Specifically, this study seeks to answer the following

questions:

3
 1. What concentration of bunker oil will allow Azolla

pinnata to survive?

2. What are the differences in the reduction of bunker oil

concentration upon treatment of Azolla pinnata R.Br.

(waterfern) when exposed;

a. gradually from two days to fourteen days

b. continuously exposed for fourteen days

Hypothesis of the Study

a. There are no significant differences in the

reduction rate of Azolla pinnata R. Br. (water fern)

in the two method of exposure.

Significance of the Study

Oil spills in different bodies of water are one of the

environmental concerns in the world. In accordance to recent

happenings, not only Filipinos will significantly benefit to

this study but also other countries.

The output of the study can be used to help a community

respond to an oil spill since Azolla pinnata R. Br. (water

fern) is readily available, easy to cultivate and

inexpensive. Also, water pollution can be decreased for a

healthier environment where ecological goods and services

would be continuous as well as economic benefits. This study

can help agencies such as the Department of Science and

Technology (DOST), Department of Health (DOH), Department of

4
Environment and Natural Resources (DENR) and the Local

Government Units (LGU) which are also concerned with the

clean-up and restoration of contaminated sites.

Aquatic animals can also benefit from this study

because oil contamination directly affects their way of life

by destroying their homes and food sources. The residents,

especially fishermen, living near bodies of water that are

also affected by oil contamination would also benefit in

this study. The research results could also provide

awareness on the importance of pollution termination and

could hopefully increase the appreciation of our youths for

an ecologically healthy surrounding for them to become more

socially responsible citizens.

This study will enable the researchers to understand

more about Azolla pinnata R. Br. (water fern). Accuracy and

precision on weighing, performing n-hexane gravimetric

analysis and other laboratory skills will be enhanced.

This study could also serve as baseline information in

the use of Azolla pinnata R. Br. (water fern) and

phytoremediation technology for oil spill in the country.

Scope and Limitations of the Study

This study is mainly concerned in the capability of

Azolla pinnata R. Br. (water fern) to reduce bunker oil

concentration in water. To determine the concentration of

5
bunker oil where most of the Azolla pinnata R. Br. (water

fern) can survive, there will 3 set ups having different oil

concentrations (0.25, 0.50 and 0.75 ml/L). The concentration

of bunker oil that will be used in determining the ability

of Azolla pinnata R. Br. (water fern) to reduce bunker oil

concentration will be based on result of the first

experiment. Gravimetric analysis for oil using n-hexane will

be conducted to determine the concentration of bunker oil

left in the water sample. The method of exposure will be

differentiated. One set up will be exposed continuously for

2 weeks and the other set up will be exposed gradually from

2 days to 14 days. Each of the set ups will have 5

replicates. This study is limited to the oil reduction

ability of Azolla pinnata. Other contaminants such as heavy

metals will not be examined. Water parameters will not be

included in this study.

Definitions of Terms

Azolla pinnata – a free floating water fern that is used in

treating contaminated soil and water

Bunker fuel - The unrefined fuel extracted from the ground

that is usually carried and spilled in the bodies of water

through oil spill

6
Continuous exposure – a kind of exposure where Azolla

pinnata R. Br. will be exposed in bunker oil concentration

for 14 consecutive days

Gradual exposure – a kind of exposure where Azolla pinnata

R. Br. will be exposed in bunker oil concentration at an

interval of two days for each set up until it reaches 14

days

Gravimetric analysis - A chemical analysis based on weighing

a final product. The mass of a product is used to calculate

the quantity of the original analyte

Heavy metals - Contaminants such as arsenic(As), iron(Fe),

copper(Cu), zinc(Zn) , lead(Pb), cadmium(Cd),

chromium(Cr),nickel(Ni) and mercury(Hg).

Hydrocarbons - An organic compound containing only carbon

and hydrogen and often occurring in petroleum, natural gas,

coal, and bitumen

Macrophytes - A macroscopic plant commonly used to

describe aquatic plant that is large enough to be seen by

the naked eye. Its ecological significance is

to provide cover for fish and acts as substrate for aquatic

invertebrates, as well as to produce oxygen and serve

as food for some fish and other wildlife.

7
Oil spill – The accidental release of oil into a body of

water presenting hazard to the environment

Phytoremediation - The use of green plants to remove or

degrade contaminants in polluted soil or water

Pollutants - Contaminants such as heavy metals, organic and

inorganic contaminants in the soil, surface waters and

groundwater that affects the natural resources and also

causes major strains in the ecosystem

Pteridophyte - Any plant belonging to the division

Pteridophyta that includes ferns, horsetails and club

mosses; plants that reproduce by means of spores and have

vascular tissue, roots, stems and leaves

8
 CHAPTER II

REVIEW OF THE RELATED LITERATURE

This literature review explores the definition, nature

and utilization of phytoremediation. The scope of this

literature review is expanded to include some of the aquatic

plants and methods used for phytoremediation. The study

investigates the capability of Azolla pinnata to reduce

bunker oil concentration in water. Cultivation methods and

factors affecting its growth are also included in this

review. This chapter also covers the composition, hazardous

effects of bunker oil as water pollutant and the recent

contamination of oil in Pasig River. Procedures on how to

measure bunker oil concentration is also stated.

Related Literature

Definition and Nature of Phytoremediation

Land, surface waters and groundwater worldwide are now

being polluted due to contaminations from industrial

activities. Toxic pollutants such as heavy metals, inorganic

and organic contaminants in the soil, surface waters and

groundwater not only affects the natural resources but also

causes major strains in the ecosystem. Interest in the use

of phytoremediation as a method of solving these

9
environmental contaminations has been growing rapidly in the

recent years of study.

Phytoremediation is a collective term of the plant

based bioremediation technologies. This refers to the use of

the green plants to clean up contaminated soil and

groundwater. The generic term phytoremediation consist of

the Greek prefix “phyto” (plant) attached to the Latin word

remediation. Phytoremediation is an attractive alternative

or complementary technology that can be used along with or,

in some cases in place of mechanical conventional clean up

treatments that are requiring high inputs, more labor and

energy intensive. It is less destructive to the environment,

cost-effective, aesthetically environmental pollutants

removal approach most suitable for developing counties. The

plants which are used in phytoremediation technique must

have a considerable capacity of metal absorption, its

accumulation and strength to decrease the treatment time of

polluted water (Gupta and Tiwari, 2011).

Phytoremediation is an emerging, cost effective, non

intrusive, aesthetically pleasing and low cost green

technology that uses plants to remove, detoxify or

immobilize environmental contaminants in growth matrix

through the natural, biological, chemical or physical

10
activities and processes of the plants. It uses the natural

ability of plants contain, sequester, remove or degrade

contaminants in contaminated area (Hettiarachchi, Nelson,

Lemunyon, Mulisa and Agudelo-Arbelaez, 2012).

Phytoremediation is a broad term used since 1991,

remediation techniques utilize specific planting

arrangements, constructed wetlands, floating plant system

and numerous other configurations. The removal of wastewater

constituents are achieved by different mechanisms like

sedimentation, filtration, chemical precipitation,

adsorption, microbial interactions and uptake of vegetation.

The principles of phytoremediation system are to clean up

contaminated water which includes identification and

implementation of efficient plants. Uptake of dissolved

nutrients and metals by the growing plants harvest and

beneficial use of the plant biomass produced from the

remediation system. The most important factor in

implementing phytoremediation is the selection of an

appropriate plant which should have high uptake of both

organic and inorganic pollutants, grow well in polluted

water and easily controlled in quantitatively propagated

dispersion. The uptake and accumulation of pollutants vary

from plant to plant and also from species to species within

a genus. The economic success of phytoremediation largely

11
depends on the photosynthetic activity and growth rate of

plants and with low to moderate. (Epps, A.V., 2009)

Phytoremediation is one of the most effective

remediation technologies for it covers a wide range of

treatment mechanisms and various contaminants. It is also

cost-competitive for it does not require the removal of

contaminant and the medium where it is contained that

requires less energy and less labor which makes it

sustainable as a remediation technology. Although the

effectiveness of technology may be dependent on the type of

the contaminant and the plant to be used, the level of

contamination, the condition of contaminated site and the

number of plants to be used. Thus, phytoremediation can be

used depending on the contaminant, situation and its

capacity on site. This technology can serve as an

alternative complimentary as a cleanup method for

contaminated sites. It can be a substitute for mechanical

and conventional clean up technologies that are often

expensive and labor intensive. More so, phytoremediation

utilizes the inherent abilities of living plants which only

uses the energy of the sun.

Like any other remediation technology, phytoremediation

has its advantages and disadvantages. Some advantages of it

12
are the following: economical since it has low maintenance

and operational cost, environment friendly because it

utilizes the natural processes of plants and aesthetically

pleasing than conventional method. It also enables

scientists to reclaim and recycle usable materials extracted

from the plant used and utilizes the inherent agronomic

benefits of plants which includes high biomass and extensive

root systems that both stabilizes the ecosystem. The

disadvantages of phytoremediation are the following: slow

speed and limited capacity and tolerance of plants on

extremely high concentrations of contaminants. Contaminants

may also be taken up by animals that fed on plants used for

phytoremediation (Alberto-Paz and Sigua, 2013).

Utilization of Phytoremediation

Phytoremediation has been gaining popularity as a

method to solve environmental contamination in recent years.

Plants that can survive and reproduce in a wide range of

environmental conditions have been utilized to clean up

broad range of contaminants including metals, radionuclide

and organic compounds found in soil and water. This green

technology involves growing plants in contaminated matrix to

remove environmental contaminants in either soil or water by

facilitating segregation and degradation of pollutants. It

13
has potential benefits in restoring a balance in stressed

environment like polluted water (Haskell, 2014).

Phytoremediation is a potential technique for the cleanup

through the metabolic and absorption capabilities of plants

as well as transport systems that can take up nutrients and

contaminants selectively from soil or water. Another

importance of plants is its ability to prevent soil erosion

and leaching that can spread the toxic substances to

neighboring areas (Maruyama, Inoue, Hasegawa, 2007; Alberto-

Paz and Sigua, 2013). It can help communities clean up the

tainted environment and restore ecological balance using

living plants (Offemaria, 2009).

This process may take several years to clean up

contaminated site that may vary according to the extent of

contamination, the area of contamination and the kind of

plant to be used (USEPA, 2012).

Aquatic Plants that are used for Phytoremediation

Freshwater as well as sea water resources are being

contaminated by various toxic elements through anthropogenic

activities and from natural resources. Therefore,

remediation of contaminated aquatic environment is as

important as terrestrial environment.

14
 Phytoremediation of toxic contaminants can be readily

achieved by aquatic macrophytes or by other small floating

plants since the process involves biosorption and

bioaccumulation of soluble and bioavailable contaminants

from water. Aquatic macrophytes treatment systems for waste-

water treatment are the need for both developed and

developing countries because they are cheaper to construct

and a little skill is required to operate. Aquatic plants

are grouped into submerged, emergent and floating-leaved

based on their leaf’s relation with water. The floating

aquatic hyper accumulating plants absorb contaminants

through its root while submerged aquatic plants accumulate

contaminants by their whole body. Floating aquatic plants

can grow in vertical and horizontal direction, thereby

increasing the photosynthetic surface area (Gupta P., Roy

and Mahindrakar, 2012; Rahman and Hasegawa, 2011).

Many researchers have used different aquatic

macrophytes like Water Hyacinth (Eichhornia crasssipes),

Water Lettuce (Pistia stratiotes), Duckweed (Lemna minor),

Esthwaite waterweed (Hydrilla verticillata) and different

species of Azolla.

Water Hyacinth has been listed as the most troublesome

weed in an aquatic system. It is a submerged aquatic plant,

15
found abundantly throughout the year in very large amount on

drainage channel system and around the fields of irrigation.

It has huge potential for removal of vast range of

pollutants from wastewater that is used to improve the

quality of water by reducing the levels of organic and

inorganic nutrients and heavy metals. The presence of its

fibrous root system and broad leaves help them to absorb

higher concentrations of heavy metals such as arsenic(As),

iron(Fe), copper(Cu), zinc(Zn) , lead(Pb), cadmium(Cd),

chromium(Cr),nickel(Ni) and mercury(Hg). Water hyacinth can

also stabilize temperature in experimental lagoons, thereby

preventing stratification and increasing mixing within the

water column. It can convert alkaline pH into neutral. This

aquatic plant can also be used in reduction of volatile

solids, phosphate, Sodium, potassium, dissolved solids and

total nitrogen, otho-P, Nitrate N, ammonia-N and chloride.

It can absorb organic substances such as phenol,

formaldehyde, formic, acetic and oxalic acetate. (Gupta P.,

Roy and Mahindrakar, 2012; Gupta R. and Tiwari, 2011; Rahman

and Hasegawa, 2011)

Water Lettuce is an aquatic plant that also grows

rapidly and is also a high biomass crop with an extensive

root system that enhances the heavy metals removal. This

plant exhibits different patterns of lead removal and can

16
even accumulate higher concentration of lead. It can remove

99.28% for lead and 65.89% for cadmium removal. It is

considered as a hyper accumulator for metals such as Zn, Ni,

As, Cr, Ag, Cu, Hg, Fe, Mg and Mn. Water lettuce are also

efficient on removal of nitrate. It is used in

phytoremediation because compared to native plants it shows

higher nutrient removal efficiency with their high nutrient

uptake capacity (Gupta P., Roy and Mahindrakar, 2012; Gupta

R. and Tiwari, 2011).

Duckweed is a fast growing and adapts easily to various

aquatic conditions. The different species of duckweed

(Lemna, Spirodela, Wolfia and Wolfiella) are distributed

worldwide in wetlands, ponds and some effluents lagoon.

Lemna minor has the capacity of improving the quality of

wastewater to an acceptable level due to its ubiquity, rapid

growth rate, ease of harvest, wide range temperature

tolerance and removing various pollutants. The efficiency of

this aquatic plant is tested by measuring some of

physicochemical characteristics of the control and plant

treatments. The highest rates of reduction are for heavy

metals for copper, cadmium, lead and zinc. Lemna minor also

removed great amount of nitrate which is attributed to its

co-existence with microorganism which play an important role

in conversion of nitrogen or plants direct up taking which

17
uses large amounts of nitrogen compounds such as NO 3,NH4

during growth period. Lemna verifies its ability to

reduction of oil and grease concentration, it reached 43%

which can be attributed to plant capability of taking

hydrocarbons and stores it in their tissues because

hydrocarbons are lipophilic pollutants as well as microbial

degradation. Reduction on temperature, pH and sulfate are

also observed but in low rates (Gupta R. and Tiwari, 2011 &

Azeez and Sabbar, 2012).

Esthwaite waterweed or Hydrilla is a submerged aquatic

weed that can grow up to the surface and form dense mats in

all bodies of water. The roots of this aquatic plant are

observed for heavy metal uptake. It has strong appetite for

both arsenic and cadmium but not so strong for lead. It also

removed metals such as Zn and Cr (Gupta and Tiwari, 2011).

Azolla is a fern frond consisting of a main stem

growing at the surface of the water, with leaves and

adventitious roots at regular intervals along stem. Azolla

are triangular or polygonal and float on the water surface

individually or in mats. It has several attributes that

merit its consideration for widespread use as an amendment

for bioaugmentation and biostimulation of contaminated

sites. Azolla filiculoides removes Fe, Zn, Mn, Co, Cd, Hg,

18
As and Ni from wastewater mixture. Azolla caroliniana based

treatment has proved as a promising tool for the treatment

of zinc at slightly higher concentration. Azolla pinnata has

been reported to accumulate high level of arsenic from

contaminated water (Deval, Mane, Joshi, Saratale, 2012; Lu,

2012; Al-Baldawi, Abdullah, Suja, Anuar and Idris, 2012).

Considerable number of literatures have been published

which described different aspects of biogeochemistry,

mechanisms and uptake of toxic elements by a large number of

aquatic macrophytes to develop effective phytoremediation

technology. Other aquatic macrophytes and some other small

aquatic floating plants that are used in remediation of

natural and wastewater contaminants are listed on the table.

TABLE 2.1 List of aquatic plants that have been tested for

the remediation of trace elements from water.

COMMON NAME OF SCIENTIFIC NAME TRACE ELEMENTS

AQUATIC PLANTS
Duckweed Lemna gibba L. As, U, Zn
Star duckweed Lemna trisulca L. Zn
Water-starwort Callitriche Cr(V)

cophocarpa
Petries starwort Callitriche As

petriei
Common reed Phragmites Cr, Cu, Ni, Pb,

19
 australis S, V, Zn, Cd
Salvinia Pb(II)

rotundifolia
Salvinia natans As, Ni, Cu,

Hg(II)
Salvinia minima As, Pb, Cd, Cr
Salvinia herzogii Cd, Cr
Eared watermoss Salvinia Zn, Hg, Cr

auriculata
Greater duckweed, Spirodela Cu, Zn, Mn,Cr Pb

intermedia
As, Hg

Spirodela

polyrhiza L.
Indian/sacred lotus Nelymbium Cr, Cu, Ba, Ti,

speciosum Co, Pb
Water spinach Ipomoea aquatica As, Cd, Pb, Hg,

Cu, Zn
Eelgrass/eelweed Vallisneria Cu, Cd, Hg Rai

spiralis L.

Elephant’s ear Colocasia Cd, Cu, Pb, Zn

esculenta
Miriophyllum Myriophyllum As

propinquum
Water lily Nymphaea violacea Cd, Cu, Pb, Zn
Marshwort Nymphoides Cd, Cu, Pb, Zn

20
 germinata
Willow smartweed Persicaria Cd, Cu, Pb, Zn

lapathifolium
Fennel pondweed Potamogeton Cd, Pb, Cr, Ni,

pectinatus Zn, Cu
Curled dock Rumex crispus Cd, Cu, Pb, Zn
Lesser Bulrush Typha angustifolia Cd, Pb, Cr, Ni,

Zn, Cu
Bulrush Typha latifolia Cr, As, Zn, Pb,

Cd, Cu. Ni
Waterweed/pondweed Elodea canadensis As, Pb, Cr, Zn,

Cu, Cd
Brazilian waterweed Veronica aquatica As
Water-milfoil, Myriophyllum Co, Cr, Cu, Pb,

spicatum Zn, Ni
Fragrant water lily Nymphaea odorata Cr
Water-milfoil Ni Myriophyllum Co, Cr, Cu, Pb,

spicatum, Zn,
Fragrant water lily Nymphaea odorata Cr
Tape grass/eel grass Vallisneria Hg

spiralis
Wild celery Vallisneria Cr

americana
Shichito matgrass Cyperus Pb, Zn, Cu, Cd

malaccensis Lam.
Water pepper Polygonum As

21
 hydropiper
Reed canary grass Phalaris Pb, Zn, Cu, Cd

arundinacea L.
Alligatorweed Althernanthera As, Pb

philoxeroides
Water lettuce Pistia stratiotes As, Cr, Pb, Ag,

Cd, Cu, Hg, Ni,

Zn
Floating pondweed Potamogeton natans Zn, Cu, Cd, Pb
Willow moss Fontinalis Cu, Zn

antipyretica
Needle spikerush Eleocharis As, In, Ag, Pb,

acicularis Cu, Cd, Zn, Sb,

Ni, Mg
Rigid hornwort Ceratophyllum As, Pb, Zn, Cu

demersum
Watercress Lepidium sativum As

L.
Watercresses Nasturtium Cu, Zn, Ni

officinale

Curly waterweed Lagarosiphon major As

Reference: (Rahman and Hasegawa, 2011) “Chemosphere”

Methods done in Phytoremediation

The use of plants as a phytoremediator differs on the

contaminants or pollutants to be remediated and the

22
processes that the plants will use upon the degradation of

the contaminant. Plants are able to do this by acting as

filters or traps through the use of their root system by

absorbing the contaminants together with the water and

nutrients that are essential for their growth (UNEP, 2014).

There are five identified types of phytoremediation; these

are Phytoextraction, Phytovolatilization, Phytodegradation,

Rhizodegradation, and Phytostabilization (Maruyama, Inoue,

Hasegawa, 2007).

Phytoextraction refers to the ability of plants to

remove metals and other compounds from the subsurface and

translocate them to the leaves or other plant tissues.

Contaminants are generally removed by harvesting the plants.

It is the best approach to remove contaminants from the

soil, sediment, and sludge but usually limited to metals and

other inorganic compounds ((Epps, A.V., 2009 and

Hettiarachchi, Nelson, Lemunyon, Mulisa and Agudelo-

Arbelaez, 2012).

Phytovolatilization is the use of plants in the uptake

of contaminants from the soil and water, transforming them

into volatilized compound and then transpiring into the

atmosphere. This process can occur with contaminants present

in soil, sediments or water and has been found to occur with

23
volatile organic compounds , including trichloroethene, as

well as inorganic chemicals that have volatile forms such as

selenium, mercury and arsenic.

Phytodegradation is the process where contaminants are

broken after they have been taken up by that plant. These

processes are observed to remediate some organic

contaminants such as chlorinated solvents, herbicide and

munitions and it can address contaminants in soil, sediments

or groundwater. (Epps, A.V., 2009)

Rhizodegradation is almost the same with

phytoextraction only that in this process it absorbs

contaminants from the water rather than the soil (United

Nations Environment Programme, 2014). It refers to the

breakdown of contaminants within the plants root zone or

rhizosphere. This mechanism is carried out by bacteria or

other microorganism whose numbers typically flourishes in

the rhizosphere. The roots provide additional surface area

for microbes to grow on and a pathway for oxygen transfer

from the environment. With its localized nature,

rhizodegradation are primarily used in contaminated soil and

in treatments of wide variety of organic chemicals which

includes petroleum hydrocarbons, polycyclic aromatic

hydrocarbons, chlorinated solvents, pesticides,

24
polychlorinated biphenyl’s, benzene, toluene, xylenes and

ethylbenzene, rhizofiltration is the use of plants,

terrestrial and aquatic, to absorb and concentrate

contaminants from polluted aqueous sources in their roots.

Rhizofiltration is typically exploited in groundwater,

surface water or wastewater and for removal of metals and

other inorganic compounds (Epps, A.V., 2009).

Phytostabilization is the process that prevents the

mobilization of contaminants through uncontaminated area by

the use of plant roots (Hettiarachchi, Nelson, Lemunyon,

Mulisa and Agudelo-Arbelaez, 2012). The use of plants to

reduce the mobility and bioavailability of pollutant in the

environment prevents their migration to groundwater or their

entry into food chain. These change the soil chemistry which

may induce absorption of contaminants into the plant root.

The physical presence of plants reduces contaminant mobility

by reducing the potential of water and wind rising (Epps,

A.V., 2009).

Hydraulic control is used to address a wide range of

contaminants in soil, sediment or groundwater through

utilization of phreatophytic trees and plants that have the

ability to transpire large volumes of water and thereby

affecting the existing balance at the site of contamination.

25
The increase in transpiration can reduce contaminant

migration from the site in groundwater plumes. (United

Nations Environment Programme, 2014).

Taxonomic Classification and Description of Azolla pinnata

Plate No. 2.1.

Sources: Mauseth, 2009; Al-Baldawi, Abdullah, Suja, Anuar

and Idris, 2012; Waseem, Preeti, Suchit and Pramood, 2012;

CABI , 2014

Generally, ferns are perennial and herbaceous. It can

be found in almost any habitat. Moist, shady forest and

lakesides are often considered as the typical fern habitat

but some species occur on dry, hot deserts like Woodsia and

Cheilanthes. Salvinia and Azolla prefers to grow floating in

water (Mauseth, 2009). The name “azolla” means that plant

dies without water (Waseem, Preeti, Suchit and Pramood,

2012).

26
 Azolla pinnata is a free-floating freshwater fern that

grows fast and is naturally found on moist soil, ditches and

marshy ponds (Waseem, Preeti, Suchit and Pramood, 2012). It

becomes especially abundant in water with high nutrient

levels, such as ponds in cattle paddocks and farm ponds

where it can completely cover the water surface. Growth

occurs all year round in tropical and sub-tropical areas

(CABI , 2014).

This freshwater fern is small, 1.5-2.5cm long and 1-2.5

cm diameter with a straight main axis, pinnately arranged

side branches progressively longer towards the base making

it triangular in shape (Invasive Species Specialist Group,

2010).

Main stem grows at the surface of the water with

alternate leaves and adventitious, fine roots at regular

intervals along the stem (Al-Baldawi, Abdullah, Suja, Anuar

and Idris, 2012). Secondary stems develop at the axil of

certain leaves (2014,

http://www.fao.org/docrep/012/i1141e /i1141e02.pdf,).

Lateral rootlets give it a feathery appearance in the water.

Leaves are small, 1-2 mm long, overlapping in two ranks. The

upper lobe can be green, brownish green or reddish and the

lower lobe is translucent brown. Short, cylindrical,

27
unicellular hairs are often present in the upper lobes

(CABI, 2014). The upper lobe is water resistant. When

submerged in water, it goes back to the surface on its right

position. When fertile, round sporocarps, 1-1.5 mm wide can

be seen on the underside at the bases of the side branches.

This aquatic pteridophyte has a very high reproduction

rate at optimum conditions wherein it doubles its biomass at

around 5-10 days. It can multiply either vegetative or via

spores (Invasive Species Specialist Group, 2010). Vegetative

reproduction is the fragmentation of fronds with doubling

possible every three days, leading to very rapid growth

rates and colonization of lakes and ponds. Sexual

reproduction leads to the formation of spores that are

released into the water. Azolla is heterosporous, a clear

adaptation to an aquatic environment. Sporangia are borne in

sporocarps, usually a pair of microsporocarps and

megasporocarps (CABI , 2014). Water fern floats on water

surface individually or in mats (2014, http://

www.fao.org/docrep/012/i1141e /i1141e02.pdf,).

Azolla pinnata has a symbiotic association with

nitrogen fixing cyanobacterium, Anabaena azollae. This alga

lives in the intercellular spaces of basal leaves of Azolla.

28
In return, heterocyst in the algal cell fixes atmospheric

nitrogen and transfers it as ammonia to Azolla.

It is locally distributed in its native range of Africa

and Madagascar, India, Southeast Asia, New Guinea and

Australia (CABI , 2014). In Asia, Azolla has been used as

green manure for crop production and a nutritional

supplement to diets for pigs and poultry. Azolla is also

applied as a controlled ecological life support system for

its strong photosynthetic oxygen-releasing capacity. It also

provides a protected environment for a fixed source of

nitrogen to the blue-green filamentous algae Anabaena

azollae (2014, http://www.fao.org/docrep/012/i1141e

/i1141e02.pdf,). Azolla pinnata is often applied to rice

fields as a nitrogen fertilizer and weed suppressant. It is

also used as a mosquito repellant and an ornament in ponds

and aquariums (Waseem, Preeti, Suchit and Pramood, 2012 and

CABI , 2014). It also has the ability to degrade hydrocarbon

in their growth medium, making it as a potential plant that

can be used in phytoremediation (Al-Baldawi, Abdullah, Suja,

Anuar and Idris, 2012).

Factors Affecting the Growth of Azolla pinnata

Azolla is an aquatic fern that floats on the surface or

water via scale-like leaves and can extend into several

29
centimeters. There are currently 6 known species of Azolla,

these are Azolla filiculoides, Azolla Mexicana, Azolla

microphylla, Azolla pinnata, Azolla rubra and Azolla

caroliniana (Sjodin, 2010).

Azolla is a plant that lives in shady areas, in normal

cases; it only needs about 50% of exposure to the sun. Tests

conducted by Controlled Ecological Life Supports Programme

have shown that this plant can grow in relatively low light

conditions. It is very sensitive to lack of water in aquatic

ecosystems such as canals, ponds, ditches, stagnant waters

and paddy fields. The doubling time of these fast growing

aquatic macrophytes is only 2-5 days. Azolla species have

various benefits but they are also considered as annoying

weeds particularly Azolla pinnata and Azolla filiculoides.

These invasive ferns can survive and reproduce in a wide

range of environmental conditions because they are more

tolerant to pollution (Sjodin, 2010).

The growth of Azolla is influenced by some abiotic and

biotic factors. Water is the main factor that affects the

growth and survival of Azolla. This fern cannot survive

without water. It should float on water surface to stay

alive (Sadeghi, Zarkami, Sabetraftar and Damme, 2012). A

strip of water not more than a few centimeters deep favors

30
growth because it provides good mineral nutrition and

reduces wind effects. Sadeghi, Zarkami, Sabetraftar and

Damme (2012) stated that Azolla prefers a medium near to

neutrality or to some extent, acidic conditions. Azolla can

survive water pH ranging from 3.5-10 and the optimum occurs

at 4.5-7.0 (2014, http://www.fao.org/docrep/012/i1141e

/i1141e02.pdf,). Temperature is another factor that

determines the growth rates of Azolla. A very high (above 30

degree Celsius) or very low temperature (below -4 degree

Celsius) could inhibit the growth of Azolla. The optimum

range for Azolla growth is between 18 and 28 degree Celsius.

Azolla pinnata’s optimum temperature range is 25-30 degree

Celsius. Light intensity is also a factor to consider in the

growth of Azolla. Photosynthetic activity, growth and

nitrogen fixation are all affected by light intensity. Low

intensities or shading had a good effect on its growth and

multiplication. They only require 25-50% full sunlight for

regular growth. Optimal light intensity for Azolla growth is

15-18 Klux and its growth and photosynthesis are inhibited

at higher intensities. It also needs enough humidity in

order to have successful growth and multiplication. The

relative humidity should be between 70 and 75% for optimum

growth. Climatic factors like the length of the growing

season and day length also regulates production of aquatic

31
plants, production summer is higher than in other seasons.

Availability of nitrogen and to some extent dissolved oxygen

are not considered as limiting factors for Azolla growth,

but growth of algae-free plants relies on combined nitrogen.

Phosphorus is an important nutrient in the successful and

rapid growth of Azolla. It will be able to grow without the

need to provide combined nitrogen such as NH4NO3 if enough

phosphorus is present in the aquatic environment. This genus

is considered to be extremely sensitive to NaCl. High

salinity level can inhibit Azolla growth. At 40 mm NaCl the

growth of Azolla pinnata was stopped. Macronutrients like

potassium, calcium and magnesium are also very important to

yield a successful and rapid growth of these species. A good

source of micro and macronutrients could be cow manure.

Biomass production of Azolla can be increased by providing

cow manure to the growth medium. Iron and trace elements

like molybdenum are important for the successful and quick

growth of Azolla, particularly in relation with its nitrogen

fixation metabolism. At lower concentrations (0.01mm), the

micronutrients showed a significant enhancement in the given

activity, whereas higher concentrations played an inhibitory

role. Phragmites sp. provides good opportunity for the

distribution of Azolla because it creates a windbreak and

shelter to this species. Insects, cephalopods, crustacean

32
and snails affect growth of Azolla by grazing on its biomass

(Sadeghi, Zarkami, Sabetraftar and Damme, 2012).

Bunker Oil and its Composition

Bunker fuel is the unrefined fuel that is extracted

from the ground. Since the oil is yet to be processed, it

can be broken down into several components, each depending

on their chemical composition and boiling point. Bunker

fuels have different types and each differs in viscosity and

can be further processed in to a wide range of petroleum

products (http//wisegeek.com/what-is-bunker-fuel.htm, 2014).

Table 2.2 Composition of Bunker C

Ingredients CAS # Percen

t
Bunker C Oil 68553-00-4 60-100
Sulfur 7704-34-9 1-5
Benzene 71-43-2 <0.1
Benzo[α]pyrene 50-32-8 <0.1
Hydrogen Sulfide 7783-06-4 <0.1
Vanadium, elemental 740-62-2 <0.1

Source: Irving Oil Refinery G.P

Aromatic compounds like benzene and benzo[α]pyrene

contribute to the immiscibility and the toxicity of the

bunker oil. These compounds irritate the mucous membranes of

33
organisms that come in contact with it. Sulfur and hydrogen

sulfide are also both toxic compounds that destroy the

breathing mechanism in animals. Vanadium, which is a heavy

metal causes poisoning and makes the oil denser, which makes

it settle in the bottom and smother the materials in the

seafloor.

Bunker oil undergoes a processing in refineries called

fractional distillation. The bunker oil is heated in the

refineries, due to the differences in the boiling points of

the different oils present in the bunker fuel they separate

when they reach their boiling point. The boiling fuels are

then siphoned out, and the heaviest oil or the residual oils

being in the bottom. Oil refineries use catalysts in order

to make the distillation process easier (HK Petroleum).

The bunker oil has very long and complex hydrocarbons,

which make it very viscous and immiscible in the water.

Being unprocessed, it also contains sulfur, hydrogen sulfide

and vanadium (refer to table 2.2) which are known pollutants

to the environment. The exact composition of the bunker oil

depends on what the source of the oil is and what refining

method is used (Irving Oil Refineries G.P).

Hazardous Effects of Bunker Oil

34
 Oil refers to a different kind of hydrocarbon based

substances that includes crude oil, petroleum products,

bunker oils, vegetable oils or other non-petroleum oils.

When oils like the crude and bunker oil is spilled into an

aquatic environment, it can harm organisms that live on that

environment by damaging parts of their food chain or

directly affecting them (Environment Protection Agency,

2014).

Oil spill may happen in several reasons. Most of the

oil spills accidents are brought up by the malfunction of

oil tanker where it leads to large amounts of oil being

released into the bodies of water. It can also be from human

activities where petroleum and other forms of oil that are

used on land are warned off into the bodies of water. Also,

through leakages when having water sports and drilling works

that is carried into the sea.

Spilled oil threatens both freshwater and marine

environment. The habitats of the animals are destroyed

during an oil spill, such as the coral reefs which serves as

the nursery of the fishes and small crustaceans (Nomack,

2010). Marshes and swamps are affected in the same way, the

mangroves in mangrove forests die because the oil that is

spilled blocks their pneumatophores, preventing gas

35
exchange. Physical smothering of heavy fuel oils which are

spilled causes the formation of slick, chemical layer that

has toxic properties and lethal effects which can cause

severe damage among organisms and ecological changes through

the loss of some key organisms from a community (ITOPF,

2014). Oil spills in coastal water affect fish and shell

fish in certain specific ways. Fish eggs and larvae that are

pelagic at or near the surface may be killed or chemically

harmed by the oil in the water. Adult fish are not usually

killed by the oil spills but the eggs that are deposited

intertidally or subtidally on the bottom are on danger

(Sindermann, 2014).

Fish that feed on planktons which are already

contaminated can subsequently accumulate high levels of

contaminants in their bodies. Larger animals in the food

chain, including bigger fish, birds and terrestrial mammals

and even humans may then consume the contaminated organism.

Based on these, bunker oil can be directly toxic to marine

organism or impact them through physical smothering,

altering metabolic and feeding rates and even their

reproduction capability (Okaloosa Oil Spill Fact Sheet,

2014).

36
 Through physical contact with oil, animals with fur and

feathers like mammals and birds lose their insulating

properties, making them vulnerable to low temperatures.

Toxic contamination through ingestion is also another effect

when the animal has ingested or inhaled the oil that has

been spilled in the bodies of water. Birds that dive to get

their food from the water will be covered in oil, this can

disable them to fly due to the oil’s heavy weight, and these

either drown or die from hypothermia. Killer whales are also

affected because it may eat fishes that are already

contaminated by the oil and this will poison them. The

blowholes of whales will be blocked and contaminated by the

oil, making them unable to breathe and die from drowning

(June 2014, http://oilspill.wordpress.com /about/).

Bunker oil has the potential to persist in the

environment long after a spill event has been detected in

sediment 30 years after a spill. Oil remains can cause

habitat loss and wildlife populations. Bunker oil leaks

affect wildlife population, making shifts on species

abundance, diversity and distribution (Okaloosa Oil Spill

Fact Sheet, 2014).

Bunker Oil Contamination in Pasig River

37
 Pasig River gives a lot of importance to Metro Manila,

Philippines since it provides food, livelihood and transport

to its residents. It also connects two major bodies of

water, Laguna Bay and Manila Bay. However, it is now

considered as the septic tank of Metro Manila due to the

large amount of waste dumped into the river. Even with the

efforts of the government to revive the quality of the Pasig

River and its tributaries, it is still continue to

deteriorate over time. The problem lies with the

insufficient funding which has resulted from weak

institutional implementation, outdated plans and

investigations and the lack of water and sewage

infrastructures. Environmental degradation and water-related

disasters have also affected the water quality of the

rivers.

Currently, all of the water ways of Metro Manila are

heavily polluted. Domestic wastes accounts for about 60% of

the total pollution in the Pasig River and the rest

originates from industrial waste (33%) such as tanneries,

textile mills, food processing plants, distilleries,

chemical and metal plants as well as solid wastes (7%)

dumped into the rivers. At the present, Metro Manila is

reportedly producing as much as 7,000 tons of trash per day

(Gorme, Maniquiz, Sung, Kim, 2010)

38
 Last June 23, 2013, thousands of liters of Bunker oil

caused panic among the residents as gas fumes wafted through

the densely populated area. The oil leak came from a

pipeline in a compound at 2657 Old Panadero St. near the

Lambingan Bridge, where the warehouse was completing the

discharge of thousands of liters of Bunker oil.

Unfortunately, warehouse workers spilled the Bunker fuel

into the Pasig River. Investigation showed the spill came

from one of the storage tanks with busted pipes. One of the

valves of the tunker tank used for transporting fuel was

left open (Burgonio, 2013).

Measurement of Bunker Oil Concentration

Bunker oil concentration is determined using the

Standard Method for the Examination of Water and Wastewater,

Section 5520B or the Extractable Hexane Gravimetric Method.

Gravimetric analysis is a chemical analysis based on

weighing a final product. The mass of a product is used to

calculate the quantity of the original analyte (Harris,

2010).

When measuring Bunker oil (HEM) gravimetrically, the

substances are extracted from the sample with n-hexane that

will be evaporated later. The residue left is weighed to

determine the concentration of oil and grease materials in

39
mg/L. To identify if reduction took place in each set-up,

the percentage reduction are calculated. It can be obtained

by dividing the difference between the initial and final

concentration and multiplying the quotient by 100 (United

States Office of Water Environment Protection Agency, 2010).

Related Studies

Local Studies

According to Estuaria and Viado (2013), duckweed

(Lemna minor) and water lettuce (Pistia stratiotes) has the

ability to reduce bunker oil concentration in water.

Although they are considered as invasive species, these

plants employ a great potential in bioremediation. Their

study compared the ability of Lemna minor and Pistia

stratiotes. L. minor and P. stratiotes were cultivated first

using water from Pasig River and was placed in plastic

containers for 3 weeks. At the experimentation, 0.5, 1 and 2

mL/L of bunker oil concentration were prepared and placed in

different plastic containers in triplicates. Ten grams of

fresh biomass of cultivated P. stratiotes and L. minor was

separately placed in different containers. A control set up

was also prepared. The laboratory procedure was maintained

under normal temperature for 2 weeks,

40
 To determine the initial and final bunker oil

concentrations, the Extractable Hexane Gravimetric Method

was used. In this method, the substances were extracted from

the sample with N-hexane and then evaporated. The

concentration of oil and grease materials in mg/L were

determined by weighing the residue left. Then the percentage

reduction was obtained by dividing the difference between

the initial and final concentration to the initial

concentration and multiplying the quotient to 100.

Researchers found out that both plants exhibit great

potential in bunker oil reduction. However, P. stratiotes

showed better oil reduction capacity in varying

concentrations compared to L. minor. The study revealed that

L. minor at 0.5, 1.0 and 2.0 mL/L initial bunker oil

concentrations have a percentage reduction of 35.34%, 34.67%

and 33.34% respectively while P. stratiotes have a

percentage reduction of 88.66%, 65.67% and 46.`7%. The

results also showed that the capacity of both plants to

reduce the bunker oil concentration decreases as the

concentration of bunker oil increases and vice versa.

Evaporation of bunker oil is proven insignificant as shown

in the control since it did not show any change in the oil

concentration. Some of the recommendations that we take in

consideration is the use of another type of aquatic plant

41
and the use of physicochemical parameters like pH and

temperature.

Abejero (2012) made a study that offers an alternative

way of treating contaminated areas with oil spills through

the use of Kapok (Ceiba pentandra) fibers packed in nylon as

an absorbent for diesel and oil in water. The use of

sorbents for oils spill cleanup is relatively acceptable but

the treatment for disposal is another concern. Different

microbes such as Bacillus megaterium, Corynebacterium

flavescens, Micrococcus luteus and Pseudomonas putida are

used to perform the degradation monitored through gas

chromatography. Sorbents are essentially inert and insoluble

materials used to remove oil and hazardous water from water

through the process of adsorption and absorption. The use of

organic such as Kapok is beneficial for it is environment

friendly, inexpensive and readily available. Even for its

disposal in contrary to the synthetic sorbents.

Bioremediation through the use of microbes even though well

researched is still an empirical technology and there are

still factors that should be well understood. The use of

microbes on bioremediation could raise concern because it

involves introduction of a new micro biota to the marine

ecosystem that could affect it and may result into

uncontrollable growth of non target organism. This

42
technology could be very useful in treatment of hazardous

substances when applied ex situ. The study could offer an

alternative way of cleaning marine oil spill through the use

of Kapok fibers packed in nylon and the employment of ex

situ bioremediation technique. The disposal of the used

kapok fibers could still pose problems and the degradation

or bioremediation of diesel oil through the use of microbes

is not assured because the laboratory and the natural marine

ecosystem is very different. Also the use of this technology

requires a lot of factors including physiochemical and

biological factors.

Foreign Studies

According to the comparative study made by Izuangbe,

Ogbeide and Olafuyi (2014) on the two aquatic macrophytes,

water hyacinth (Eichhornia crassipes) and water lettuce

(Pistias stratiotes) as a remediator for the produced water,

it led to the conclusion that water hyacinth is a better

candidate than water lettuce. The study used the water

collected in the detention pits to be analyzed through

different experimental set up. The aquatic plants which were

grown in fresh water ponds were removed from the ponds and

introduced into each experimental pond containing

approximately 250 liters of the produced water. Data

43
obtained from this six weeks study shows that using water

hyacinth and water lettuce improved water quality by

decreasing total solids. However, water turbidity value

increased due to deposition of dead organic matter and

growth of algae. Growth of water hyacinth and water lettuce

was limited. High salinity levels appear to be the principal

reason for inhibited growth, along with the presence of

uncharacterized soluble compounds making up a significant

fraction of the produced water soluble chemical oxygen

demand. It is worthy however to note that among the nineteen

parameters analyzed in this study, water hyacinth was able

to reduce fourteen parameters while water lettuce reduced

three parameters. Some of these parameters are the contents

of coliform, analysis of sulphate, pH, salinity, turbidity,

oil and grease content, electrical conductivity and

dissolved oxygen. The above comparative study has led to the

conclusion that water hyacinth is a better candidate than

water lettuce in the remediation of produced water.

Loveson, Siralingam and Syamkumar (2013) conducted an

experiment on Spirodela polyrhiza as a phytoremediation

agent. Collected wetland water had undergone a sieving setup

in order to remove the large suspended solids in the soil.

The wetland was immediately transferred to aquarium set-up,

there were 4 aquariums used. Each aquarium had a length of

44
18 inches and had a depth of 10 inches and 9 inches wide.

Duckweed used for the experiment was collected from an

unpolluted pond near Fort Kochi, Kerala. The macrophytes

were washed with tap water. Approximately 50g of fresh and

wet duckweed was placed in three aquariums, each of the

aquariums were filled with polluted wetland diluted with

water at 1:4 ratio. The control in the experiment was the

aquarium that had the same amount of the diluted wetland

water and distilled water but has no duckweed. The

experiment was conducted under laboratory conditions having

a temperature of 25oC and exposure to lighting with a ratio

of 8 light: 16 dark. The detention time for the duckweed in

the experiment was 8 days in the first set-up, 4 days in the

second set-up and 2 days in the third one. The study

employed the duckweed as an effective plant for

phytoremediation from the polluted wetland water. The

experiment has shown high metal removal rates close to 100%

have been seen in the wetlands set-up. It is an easy and

cost-effective alternative for the treatment of wastewater.

Azeez and Sabbar (2012) studied the efficiency of

duckweed Lemna minor in Phytotreatment of Wastewater

Pollutants from Basrah Oil Refinery. In their set-up, 20L of

Basrah oil refinery wastewater was used. The water was

diluted with distilled water with a ratio of 1:4. The

45
duckweed used for the experiment was collected from the

Basrah University and were washed with tap water and by

distilled water afterwards. A 100g of fresh biomass of

duckweed was added in the three aquaria, the fourth one

having no duckweed, served as the control of the experiment.

The set-up was under laboratory conditions, having a

temperature of 25oC and lighting ratio of 8 light: 16 dark.

Physiochemical analysis of the samples were taken every 7

days and repeated about 4 times a month. The results of the

experiment has shown that there is a significant decrease in

the levels of oil and grease with a reduction rate of 43.4%

and metals like Pb were reduced by 98.7%, Cu by 99.8%, Zn by

72% and Cd by 99.6%.

Al-Baldawi, Abdullah, Suja, Anuar and Idris (2012)

conducted a test to investigate the ability of small water

fern Azolla pinnata to survive when exposed to diesel

contaminants. Diesel is a toxic substance in the environment

and causes a great threat to the ecosystem and human being.

Its toxicity is due to the presence of aromatic hydrocarbons

such as benzene, toluene, ethylbenzene and xylenes. It was

found out in the study that an increase in the concentration

of diesel, withered plants also increases. At the end of 10

days, 20% of Azolla pinnata were withered at lower

concentration of 0.5 ml/L and 50% withered at concentration

46
of 1 ml/L. It was concluded that the concentration of diesel

must not exceed 0.5 ml/L (v/v) in the phytotoxicity test

because 50% of withered plants means that plants cannot

survive and diesel concentration was too high for this

species.

An experiment conducted by Lu (2012) with a pond

containing a water hyacinth was reported to remove Ortho-P,

Nitrate N, and ammonia-N by 99%. Small tank polycultures of

duckweed species, Lemna minor and Spirodela polyrhiza are

found to remove 404 mg N m-2 and 84 mg P m-2 from dairy barn

wastewater. Phosphorus removal rate of 60-92.2% are achieved

in wastewater system through utilization of Lemna gibba. Two

species of Azolla, Azolla filiculoides and Azolla pinnata,

removes N from mixed wastewater resulting in more than 50%

decrease in concentration. Typha latifolia and Scirpus

lacustris has removal rates of NH4-N by over 85%. Salvinia

herzogii efficiently removes Cd from water and Salvinia

minima have been reported as a hyper accumulator of Cd and

Pb with bioconcentration factors. Azolla filiculoides

removes 91% Fe, 41.5% Zn, 82.5% Cu, 37.7% Mn, 12.1% Co,

46.7% Cd and 67.29% Ni from wastewater mixture and Azolla

pinnata removes 92.7% Fe, 83% Zn, 59.1% Cu, 65.1% Mn, 95%

Co, 90% Cd, and 73.1% Ni.

Analysis of Review of Related Literature

47
 This review of related literature revealed that aquatic

macrophytes and other small floating plants have the

capability to contain, sequester, remove or degrade

contaminants in contaminated area. Phytoremediation has been

effective in cleaning up heavy metals, inorganic and organic

compounds. Water hyacinth (Eichhornia crasssipes), water

lettuce (Pistia stratiotes), duckweed (Lemna minor) and

different species of Azolla are the common aquatic plants

used in phytoremediation. However, the previous studies are

limited in the ability of the said plants to uptake heavy

metals such as lead, arsenic, cadmium, iron, copper and

mercury. Studies about the degradation of oil and grease

using aquatic plants is not yet fully known and

experimented.

Oil spill incidents are one of the major environmental

problems in our society. Recently, thousands of liters of

bunker oil leaked in the Pasig River, threatening to cause

pollution and massive environmental damage. In response to

this, a study that tests the ability of water lettuce and

duckweed in reducing concentrations of bunker oil was

conducted. This study aimed to test the said potentiality of

other aquatic plants as phytoremediator in degrading oil

contaminants in water. The study revealed that both plants

exhibit great potential in bunker oil reduction. However,

48
water lettuce showed higher capability for reduction in

varying concentrations of bunker oil compared with duckweed.

Despite of the effectiveness of water lettuce, it tends to

clog on waterways because of its size.

In this research, Azolla pinnata will be utilized to

reduce bunker oil concentrations. Compared to other aquatic

plants like Pistia stratiotes (water lettuce) and Lemna

minor (duckweed), Azolla pinnata R. Br. (water fern)

possesses more of the properties of an ideal plant for the

use in phytoremediation such as fast growth rate, nitrogen

fixing ability, high biomass production, moderately

extensive root system, easy to harvest and high tolerance to

a wide range of heavy metals (Sood, Uniyal, Prasanna and

Ahluwalia, 2012, http://www.ncbi.nlm.nih.gov/pmc/

articles /PMC3357840/). In addition, Azolla pinnata R. Br.

(water fern) is smaller than Pistia stratiotes (water

lettuce) and Lemna minor (duckweed) thus reducing its

tendency to clog on waterways. Based on the studies done by

Deval, Lu, Al-Baldawi and Abdullah in 2012, Azolla pinnata

proves its capacity in removing heavy metals and its ability

to degrade hydrocarbon in their growth medium. Azolla

pinnata is a fast growing fresh water fern that is

considered as invasive species. It can survive and reproduce

49
in a wide range of environmental conditions because they are

more tolerant to pollution.

This study can be used to help a community or even

countries respond to an oil spill since Azolla pinnata is

readily available, easy to cultivate and inexpensive.

Organisms like aquatic animals and residents living near

bodies of water affected by oil spills will benefit in this

study. This study could also serve as baseline information

in the use of Azolla pinnata and phytoremediation technology

for oil spill in the country.

50