Wastewater Treatment Using Constructed Wetlands Through

Phytoremediation

Requirements for the subject

Res 313: Research Method
During 1st Semester 2020-2021

Bachelor of Science in Civil Engineering - 3A

School of Engineering
Biliran Province State University

Student: Jake Nathaniel R. Abad

Professor: Buhn Alay G. Colorado

105
 CHAPTER 1
INTRODUCTION

1.1 Background of the Study

Constructed wetlands are human engineered systems that utilize natural

treatment processes to reduce the pollution levels in wastewater. The

combination of soil, plants and microorganisms efficiently remove organic

pollutants, nutrient concentrations and toxic contaminants in water using a variety

of physical, biological, and chemical processes. The energy that driven all these

processes is provided mainly by the sun. The research on the capacity of marsh

plants in the reduction of organic pollutants and nutrients in aquatic systems

started in the 1950’s in Germany. Since then, various designs of constructed

wetland have been developed. Thousands of facilities as an outcome of such

investigation are currently in use in Europe, Australia, and the United States.

Recent years have seen the rapid proliferation of constructed wetland systems in

Africa and Asia. There are two basic types of constructed wetlands, surface flow

and subsurface flow. Surface flow wetlands are essentially shallow ponds

planted with floating and emergent species of aquatic weeds. The water flow in

these systems is observed almost entirely above the soil layer. The flow in

subsurface flow in the wetlands occurs under the surface and within the soil

planted with emergent aquatic plant species. Subsurface flow is either horizontal

(the water moving parallel to the surface) or vertical (the influent is distributed

across the surface). It percolates down the media and can be collected in a
bottom layer. Subsurface wetlands are generally more efficient. These require

less land area (Kuchta, and Sarana, 2008).

Major water bodies of all over the world are mostly polluted by the

discharge of domestic sewage, trade effluents and industrial wastewater. But it is

needed to be treated through low cost, an ecofriendly technology for sustainable

future. The efforts can be made to treat the wastewater using suitable aquatic

weeds by using rootzone technology (Metcalf and Eddy, 1991; Chavan et al.,

2009a). Such efforts have been made in the present investigation using various

aquatic plants in constructed wetland.

Phytoremediation technology in the form of constructed wetland or natural

water marsh is a nature’s gift for control of water pollution and for sustaining

livelihood. Wetlands, both natural and constructed can purify wastewater due to

their ability to degrade, absorb or filter the pollutants and to take-up nutrients

from the water or wastewater. Therefore, the use of constructed wetlands for

wastewater treatment is becoming more and more popular. The wetlands are

found across the country and around the world due to their diverse applications.

The constructed wetland treatment systems can be established almost

anywhere, including on lands with limited alternative uses (USEPA, 1988). They

can often be an environmentally acceptable, cost-effective treatment options,

particularly for small communities. The available water resources are adequate

for the present community in some areas based on natural average consumption

but marginal in other areas such as arid, semi-arid regions to meet their routine

water needs. The wastewater from various sources can be efficiently treated by
constructed wetlands and reused. There is vast literature available on this

subject. Most of it has been devoted for the fulfillment of the gap that exists

between the generalized approach and the local situations (Scierup et al., 1990;

Schreijer et al., 1997; Newman et al., 1998; Dilshad et al., 2010). This gap

provides a wide scope to undertake research to develop an efficient system and

evolve and establish an effective procedure to fulfill the research gap between

the literature information and localized need for adaptive aquatic weeds for the

phytoremediation. The efforts have been made to contribute to fill up this gap

with practical efforts in present work using Water Hyacinth.

The root zone process is the natural remedy to the wastewater pollution

problem. Wetland based technology is an ecofriendly, self–contained, artificially

engineered constructed wetland ecosystem. In this process the wastewater can

flow laterally through specially prepared impervious bed in which specific wetland

plants are grown. The root zone process has been fully commercialized to treat

industrial and domestic effluents using specific aquatic macrophytes for the

control of water pollution (Brix and Schierup, 1989; Dhote., 2007). One of the

integrated components of this process is the need of adaptive and efficient

aquatic weeds. The plants grow in the wastewater by absorbing nutrients at

faster rate turning these weeds to a desirable productive use. The plants hold

themselves in the interporous molecules of soil layer or support system through

their roots and rhizomes. These form an intricate network of underground stem.

The roots of these plants grow rapidly and provide air passages through the

sludge. In turn, the sludge provides a host area for many biological communities
to colonize, develop and continue to mineralize the sludge contents. It helps to

optimize the microbiological, chemical, and physical processes naturally

occurring in the wetland (Bates and Hentages, 1976; Hammer, 1989).

The wastewater is made to flow horizontally along a seated path of porous

bed reactor where oxygen is introduced biologically via helophytes. The porous

bed material provides large surface area for the colonization or adsorption of

bacteria which degrades organic load (Brix, 1994). In the densely rooted bed, the

activity of microorganisms increases in terms of both, quality, and quantity. The

large contact area between water and the bed particles allow eliminating nitrates

and phosphates by both processes, absorption, and chemical precipitation. The

aerobic and anaerobic bacteria carry out active role in the reduction of COD and

BOD of wastewater and help to reduce the extent of groundwater contamination.

This technique is suitable for the sustainable wastewater and wastewater

management. It may be preferred for recharging of groundwater (Rao and

Mamatha, 2004; Vymazal, 2009). In the Philippines, almost all the water bodies

had been occupied with various types of macrophytes. These may be free

floating, submerged, and emergent (shoreline) plants are an integral part of the

aquatic ecosystem and act as bio-filters. The conventional wastewater treatment

process is inconvenient in the form of its operation and very costly for its

maintenance. Therefore, efforts are made by various researchers for the use of

natural devices, which can be used as an eco-friendly and effective source of

treatment. (Dhote et al., 2007; Chavan et al., 2012a, 2012b).

 The present studies are aimed to investigate the phyto-treatability

potential of Water Hyacinth. The investigations are carried out to determine the

potentiality of Water Hyacinth at different concentrations of pollutants for

reducing the water pollution in the study region. Considering the abundant

availability of Water Hyacinth in the Philippines, this plant has been selected for

the present study. This plant is locally adaptive, abundant, fast growing and

easily available which made it easy to select for the present investigation. In this

work, wastewater was treated in different concentrations of pollution load in

Angular Horizontal Subsurface Flow (AHSSF) constructed wetland using Water

Hyacinth.

1.2 Statement of the Problem

This study entitled “Wastewater treatment using constructed wetlands

through phytoremediation” will answer the following objectives generally:

To assess the heavy metal removal capacity from specific plant species on

Copper and Chromium through phytoremediation.

Specifically, it aims to answer the following:

1. What is the effect of heavy metals (Co and Cr) in the biomass of water

cabbage (Pistia Stratiotes), Coontail Duckweed (Ceratophyllum

Demersum), and Water Hyacinth?

 2. Which among the water cabbage (Pistia Stratiotes), Coontail Duckweed

(Ceratophyllum Demersum), and Water Hyacinth has the ability to reduce

heavy metals?

3. What is the maximum accumulation potential of water cabbage (Pistia

Stratiotes), Coontail Duckweed (Ceratophyllum Demersum), and Water

Hyacinth on aqueous solution of the potassium dichromate and copper

sulfate?

1.2.1 Null Hypothesis:

The water cabbage (Pistia Stratiotes), Coontail Duckweed (Ceratophyllum

Demersum), and Water Hyacinth has the ability to remove heavy metals from

polluted water.

1.2.2 Alternative Hypothesis:

Hypo1 – 20g of water cabbage (Pistia Stratiotes), Coontail Duckweed

(Ceratophyllum Demersum), and Water Hyacinth is the best for the natural

approach in phytoremediation of chromium and copper.

Hypo2 – Water Hyacinth is the best among the 3 plants in removing heavy

metals from soils and water in different concentrations of aqueous solution of

potassium dichromate and copper sulphate.

1.3 Purpose of the Study

The purpose of this study is to find a cost-effective technology for

combined wastewater treatment by using different plants and algae in CWs. The

lab scale information obtained from the current research will help to design CWs

at large scale.

1.4 Significance of the Study

This study is important because it will reveal the natural ability of Pistia

Stratiotes, Ceratophyllum Demersum, and Water Hyacinth in bioaccumulating or

rendering harmless contaminants in water. Likewise, this project could give

people an idea on how to use Pistia Stratiotes in medicine for the treatment of

different external and internal diseases. Furthermore, this will also add

knowledge to the next researchers of what other species of Pistia Stratiotes,

Ceratophyllum Demersum, and Water Hyacinth can be used in phytoremediation

of heavy metals. Lastly, this study aims to contribute to the field of science and

technology in such a way of determining a sustainable approach in degrading

heavy metals in bodies of water.

1.5 Limitations and Delimitations of the Study

This study entitled “Wastewater treatment using constructed wetlands

through phytoremediation” focuses mainly on the ability and effectiveness of

specific plant species like Pistia stratiotes, Ceratophyllum demersum, and Water

Hyacinth in removing heavy metals in polluted water particularly copper and

chromium.

A study like this was conducted at Bayorbor, Mataasnakahoy, Batangas.

Considering that the scope and limitation then the result should be tested or

validated in other areas in order to prove if it can be duplicated.

1.5.1 Limitations of the Study

This study is somewhat held back or limited by several factors. (1) The

depth of the treatment zone is determined by plants used in phytoremediation. In

most cases, it is limited to shallow soils. (2) High concentrations of hazardous

materials can be toxic to plants which means it might not work depending on the

plant. (3) It involves the same mass transfer limitations as other biotreatments.

(4) It may be seasonal for the plant type and species, depending on location. (5)

It can transfer contamination across media, e.g., from soil to air. (6) It is not

effective for strongly sorbed (e.g., Polychlorinated biphenyls/ PCBs) and weakly

sorbed contaminants. (7) The toxicity and bioavailability of biodegradation

products is not always known or have not been studied on well enough. (8)

Products of this treatment may be mobilized into ground water or bioaccumulated

in animals which is a problem. (9) It is also still in the demonstration stage.

1.5.2 Delimitations of the Study

The main environmental concerns caused by the industrial sector are

deeper, more complex issues of polluted ground water and land contamination
which can also lead to unsuitable air quality. These more complex issues are not

addressed in this part of the study, but primarily serve as an

introduction/background to the severity of the industrial pollution and are included

in the urban framework. Therefore, this dissertation does not attempt to solve the

general problem of industrial pollution but instead, the project focuses on a facet

of the greater water issue which is that of untreated industrial effluent. This study

will only attempt to figure out another more environmentally friendly process by

which the sewage is treated without just segregating the rest of its toxic content.

The research will focus on the removal of heavy metals with the

phytoremediation wastewater treatment to ensure that they are permanently

removed from the environment and not disposed of as hazardous waste which

usually have no proper place to be disposed into. This study could try to use all

types of plant species that can work in phytoremediation, but it will have to

depend on their location and other uses from which these plants have. That’s

why only specific types of plants that can help eliminate the heavy metals from

the sewage water and also be used in agriculture will be chosen (e.g.,

Pistia stratiotes/water cabbage).

1.6 Theoretical Framework

Research question: Can Phytoremediation help eliminate the toxic heavy metals

in Wastewater?

Sub-Questions:
 1. What are the physical and chemical characteristics of the soils used in the

phytoremediation process?

2. How much heavy metal content is there from the wastewater in the soil of

the Phytoremediation process?

3. Is there a method for plants to absorb these heavy metals or increase their

absorption?

Phytoextraction of heavy metals with Canola in model field

They performed the phytoextraction with canola in the model fields to estimate

ant effectiveness.

1.6.1 Experimental conditions

The experiment is conducted on the research fields of Faculty of

Agriculture, Namık Kemal University, Turkey, according to the randomized block

design with 3 replicates. 100 mg/kg Co, Cr, Ni and Pb ions are taken from

CoSO4, Cr (NO3)3, NiSO4 and Pb (NO3)2 compounds and applied to the soil as

contaminants. There are 51 parcels with 4 contaminants (CoSO 4, Cr (NO3)3,

NiSO4 and Pb (NO3)2) × 4 chelate doses (EDTA) (0, 5, 10 and 15 mmol/kg) × 3

replicates + 3 control. The control parcels in which the contaminants and chelate

are not applied are organized as three replicates. Each parcel in the test are

sized as (3 × 1.2 m): 3.6 m 2 including four rows (for instance for lead element

PbEDTA0, PbEDTA5, PbEDTA10 and PbEDTA15). The row distance in each parcel is 30 cm,

the distance between each parcel is 0.5 m and between each block is 1.5 m. The
height of each block is 3 m and the width are 31.2 m (there are 4 parcels in each

block). Therefore, a whole block is of 93.6 m 2 and the total test area is of 284.6

m2, including the distances between the blocks (Figure 1).

Figure 1. Different views from experiment fields (original).

1.6.2 Physical and chemical characteristics of the test soils

The physical and chemical characteristics of the samples taken from the

testing field are presented in Table 1.1. According to the table, the soil’s pH is

neutral, low lime and has insufficient organic matter. Its available phosphorus

content is enough as well as the exchangeable potassium. The amount of

available iron is average, available copper and manganese is enough, and

available zinc is insufficient. Also, the testing soil is classified as clay in terms of

texture [21, 22].

Soil properties Unit Values

pH (soil: water=1:2.5) 6.81
EC (x106) dS/m 128.3
CaCO3 % 2.40
Soil properties Unit Values
Organic matter % 1.88
P2O5 kg/da 11.42
K2O kg/da 25.32
Fe mg/kg 3.46
Cu mg/kg 0.63
Zn mg/kg 0.40
Mn mg/kg 5.72
Clay % 42.98
Silt % 25.44
Sand % 31.58
Texture class C
Table 1.1.
Some physical and chemical characteristics of the testing soil [23].

1.6.3 The amount of heavy metal (Cr, Co, Ni and Pb) before the test and

after the incubation

The extractable Cr, Co, Ni and Pb contents of the testing fields are

identified before the heavy metals are applied to the soil and they are presented

in Table 1.2. 100 mg/kg Cr, Co, Ni and Pb are applied to the testing field and left

to incubation for a month. The extractable heavy metal contents are determined

after the incubation. T-test is applied to the results and the standard error values

are given in Table 1.2.

Heavy metal Before polluting After polluting

Cr 0.10 ± 0.01** 5.80 ± 0.07**
Co 0.08 ± 0.05** 2.25 ± 0.02**
Heavy metal Before polluting After polluting

Ni 0.95 ± 0.05** 6.20 ± 0.13**

Pb 0.93 ± 0.01** 7.52 ± 0.04**
Table 1.2.

The extractable heavy metal contents before and after the application of the

contaminants to the testing soil.

**

p< 0.01.

When the Table 1.2 is examined, it can be observed that the amount of

extractable heavy metal contents before the test is acceptable and does not have

any contaminant characteristic [1]. A remarkable increase is observed as a result

of 100 mg/kg Cr, Co, Ni, Pb heavy metal application upon one-month incubation.

These increases are determined as 1% significant statistically.

1.6.4The effects of EDTA applications on heavy metal contents (Cr, Co, Ni,

Pb) of the root and shoot of the plant

The effects of increasing doses of EDTA applications on heavy metal contents

(Cr, Co, Ni, Pb) of the root and shoot of the plant are presented in Table 1.3.

Canola Chrome (Cr) Cobalt (Co)

EDTA application Root Shoot Root Shoot
Control 2.75 ± 0.57a 4.23 ± 0.58a 1.95 ± 0.02a 2.18 ± 0.04a
0 mmol/kg 14.70 ± 007b 8.25 ± 0.56a 13.30 ± 0.08b 11.56 ± 1.39b
5 mmol/kg 29.16 ± 0.58c 26.42 ± 0.21b 28.60 ± 1.50c 24.45 ± 0.59c
10 mmol/kg 55.12 ± 0.99d 40.45 ± 1.05c 51.40 ± 2.61d 39.12 ± 1.14d
15 mmol/kg 70.50 ± 1.92e 52.20 ± 1.50d 75.40 ± 1.56e 45.20 ± 0.60e
Nickel (Ni) Lead (Pb)
EDTA application Root Shoot Root Shoot
Control 4.76 ± 0.11a 5.83 ± 0.63a 3.78 ± 0.45a 4.96 ± 0.56a
0 mmol/kg 24.43 ± 0.13b 13.12 ± 0.97b 24.50 ± 0.97b 18.90 ± 0.54b
5 mmol/kg 49.65 ± 0.34c 37.60 ± 0.56c 41.40 ± 0.66c 35.20 ± 0.05c
10 mmol/kg 77.80 ± 0.60d 61.40 ± 0.28d 87.80 ± 0.90d 63.14 ± 1.14d
15 mmol/kg 85.30 ± 1.01e 65.10 ± 0.057e 95.40 ± 0.17e 70.12 ± 0.01e

Table 1.3.

The effects of EDTA applications on the amount of heavy metals (Cr, Co,

Ni and Pb) in the roots and shoot of canola plant that were grew* (mg/kg)

[23, 24, 25, 26].

Each heavy metal element, root and shoot is examined separately with

three replicates.

The amount of Cr in the roots and shoot of the canola plant which grows

on Cr-applied fields rapidly increased after 0 mmol/kg EDTA dose and reached

the highest level with 15 mmol/kg EDTA dose. These increases were determined

as 1% significant statistically.
Like the Chrome element, the amount of cobalt in the root and shoot of canola

plant which grows on the cobalt-polluted field increased with EDTA applications.

These increases were determined as 1% significant statistically (Table 1.3).

The amount of nickel in canola plant which grows on the field that has

been polluted with nickel increased with EDTA applications, and the highest

levels were achieved on the parcels which were applied with 15 mmol/kg EDTA

dose. It can be concluded that with the increasing doses of EDTA, the Ni

concentration in the roots and shoot of the plant has also increased. These

increases were determined as 1% significant statistically and various groups are

formed in Duncan multiple comparison test (Table 1.3).

The amount of Pb in canola plant which grows on the field that has been

polluted with Pb increased with EDTA applications, and the highest levels were

achieved on the parcels which were applied with 15 mmol/kg EDTA dose. It can

be concluded that with the increasing doses of EDTA, the Pb concentration in the

roots and shoot of the plant has also increased. These increases were

determined as 1% significant statistically and various groups are formed in

Duncan multiple comparison test (Table 1.3).

In Table 1.3, the amount of heavy metals (Cr, Co, Ni and Pb) in canola

plant’s roots and shoot, Duncan multiple comparison test and standard error

values are presented. According to Table 1.3, the amount of heavy metals in the

roots is higher than the heavy metals in shoot.

 According to Table 1.3, on the fields which are applied with 0 mmol/kg

EDTA doses, the amount of Cr, Co, Ni and Pb heavy metals in the roots and

shoot of the canola plant is lowest and the highest heavy metal level can be

detected in 15 mmol/kg EDTA dose. The results are equal to other research

results on this subject. The researchers have explained that the solubility and

absorption of the heavy metals get easier for the plant with the increasing doses

of EDTA application [27, 28, 29, 30, 31].

According to the results of this research, EDTA applications should be

conducted by growing hyper-accumulator plants on the soils in order to decrease

Cr, Co, Ni and Pb heavy metal contamination under the toxicity levels. Because,

it has been proved that with the increasing doses of EDTA application some

heavy metals (Cr, Co, Ni and Pb) can be removed from soil with

phytoremediation (Phytoextraction) method.

1.7 Why farmers prefer wastewater despite the risks? The big question

It has been shown that wastewater use can contribute to improved

livelihoods at the household level (Raschid-Sally et al., 2005), particularly

through recycling of water for irrigation and specifically with additional direct

benefits of nutrients for plant use (Janssen et al., 2005). This noble approach of

agricultural use of domestic wastewater, if well designed and managed, has

the potential to address the problems of local water shortages, improved soil

fertility and can also be viewed as part of a treatment system to reduce

environmental pollution (Jime´nez, 2005). The handling of wastewater to reduce

the pollutants can be direct through initiatives of municipalities (e.g. use of

chlorine or UV radiation) that kill pathogens or indirectly by informed choice of

hyperaccumulator crops that can extract heavy metals from water or soils

along the water channels and partially by passing the wastewater through

oxidation ponds (an oxidation pond is such a pond that contains partially treated

wastewater which is then left to allow the growth of algae and bacteria

which decompose the rest of waste). Generally, farmers in urban and peri-urban

areas of nearly all developing countries who need water for irrigation often have

no other choice than to use of wastewater. They even deliberately use undiluted

wastewater (a case where the government has taken intervention to partially

treat the wastewater) as it provides nutrients or is more reliable or cheaper than

other water sources (Keraita and Drechsel, 2004; Scott et al., 2004). This kind of

unprecedented response creates daunting challenge to policy makers in trying to

formulate policy on treatment or partial treatment of wastewater in their planning

framework for wastewater use or re- use. In Faisalabad for instance, report

indicates that farmers refuse to use treated wastewater from oxidation ponds

because the lower nutrient values require additional fertilizer inputs (Qadir et al.,

2008). Comparative studies between wastewater and non-wastewater farmers

have shown that the former make more income not only from savings in

fertilizer but additionally the reliable wastewater supply allows them to grow

short-cycle cash crops (van der Hoek 2004; van der Hoek et al., 2002; Ensink

et al., 2004; Karanja et al., 2010). Wastewater may be used directly or after

mixing with sewage channeled into natural drainage systems, from where the

polluted water is used for farming (Qadir et al., 2010). Most commonly, a year-
round vegetable production is practiced, for which farmers have a good market.

In many places in the world, this form of production has great importance as a

source of income and livelihood for many people. Huibers and Raschid-Sally

(2005) observed that farmers usually have no land rights and make use of

available urban land belonging to property owners or the state, until they are

thrown out. Consequently, there is no irrigation infrastructure and no means for

regulation and control. Watering of the plants is done by simple means, for

example using buckets or watering cans. This practice leads both to health risks

to the irrigators who are in close contact with the polluted water, and there is also

high risk of crop contamination. Interestingly crop contamination may also occur

during the crop handling after harvest (Amoah et al., 2007), and this applies

irrespective of whether the crop is produced with wastewater or other source of

water.

1.7.1 The drive, challenges, and prospects: Considerations for policy

makers

According to Huibers and Raschid-Sally (2005), wastewater irrigation

schemes represent an example of planned and institutionalized use of

wastewater in the downstream rural areas of big cities. This wastewater is rarely

treated at secondary level, and in many cases the quality of the effluent is below

standard because of poor performance of the treatment system (WHO 2006).

Under these schemes, an effort is made for an optimal recycling of both water

and nutrients. Huiber and Raschid-Sally (2005) argue that since these schemes

are formal, government rules on crop choice and management are strict and
much more controlled than in the other situations as we have tried to

schematically present in Fig. 1.1. The involvement of authority in wastewater

treatment more than often restricts farmers from growing the crops of their choice

suitable to the market, or they are forced to apply expensive management

systems. Theoretically, as observed by Qadir et al (2007), farmers could

compensate income loss due to crop restrictions by the lower water price they

would normally pay and by saving on fertilizers. However, in practice farmers

have insufficient insight into the nutrient composition in the water they use and

keep applying chemical fertilizer as well (this is scenario where is wastewater is

fully or partially treated). This brings unnecessary costs to the farmer, while the

resulting over-fertilization is a source of further pollution (Qadir et al., 2010). At

present, to our knowledge, there are no microbiological irrigation water quality

standards that acknowledge the concept of an acceptable level of health risk for

irrigators and the wider community, other than zero risk. In the absence of other

norms, the WHO microbiological quality guidelines for the design of wastewater

treatment plants, where the effluent is intended to be used for irrigation, are used

extensively to evaluate the health risks arising from the use of polluted water

sources for irrigation (WHO, 1989). Many developing countries are clearly

below this threshold. In Ghana, for example, only 7 out of 44 smaller treatment

plants are functional and probably none meets the designed effluent standards

(Obuobie et al., 2006). Whereas this is pertinent, the public officials must

consider potential impacts on the poor when designing policies and programs

since stringent adherence to the requirement may deter the informal farmers to
access the wastewater resources profitably. And this is therefore one amongst

the numerous challenges that affect peri-urban informal farmers relying on

wastewater. The other greatest challenge might be ensuring that low-income

residents of peri-urban and rural areas who rely on polluted streams or

wastewater for crop production are not deprived of their livelihoods. Many poor

farmers have been using these water sources for years without formal water

rights and a study shows that banning the use of polluted water was estimated

to affect about 12,700 households or 90,000 people living around the city of

Kumasi by late 90s, depending on dry-season irrigation (Cornish and

Lawrence, 2001). Such predicaments faced by farmers in Kumasi and Accra are

very similar to those of their counterparts in Nairobi, particularly in Ruai and Maili

Saba (Cornish and Kielen 2004; Karanja et al., 2010). The other challenge is the

accumulation of heavy metals and microbial contamination of crops, particularly

vegetables produced by such system. Information on the pattern and

distribution of heavy metals, accumulation and translocation in the edible plants

from the sites of deposition in agricultural field receiving industrial wastewater

would be helpful for the selection of suitable plant species that can be used

as accumulator plant to minimize the concentration of these metals in the highly

polluted soils (Salt et al., 1995; Rai et al., 1996; McGrath, 1998; Sinha et al.,

2002). The kind of the information needed to be shared by all stakeholders is

proposed in the current review as indicated in Fig. 1.1. As has been mentioned

here, the metal accumulation in the various parts of the plant depends upon

availability and species of metals in soil, solubility, their translocation potential

and the type of plant species. The bioavailability of trace metals in soils also

governed predominantly by various soil properties (pH, redox potential, cation

exchange capacity and organic matter) (Karanja et al., 2010). And this is of

significance to technical advisors (e.g. agricultural officers) to ensure such soil

properties are amended to alleviate inducing availability in the soil media for

subsequent uptake by crops. The other important concern to be observed in

sustainable wastewater use is the inclusion of user and consumer health

protection concerns, through interventions at farm level, post-harvest measures,

public policies to motivate better management of wastewater. This can be

through ensuring that the farmers wear gloves, are frequently medically

examined and research come up with a detergent that farmers use to clean the

vegetables under supervision of experts before being ferried to market. Vinegar

can be a good option.

So far, the results of most researchers have ignored other routes of metal

pollutants into plant tissues. We speculate that there may be substantial amount

that get to plants as aerial droplets and this need to be separated from plant

uptake from soil. To separate this, a greenhouse experiment using contaminated

soils or using wastewater may be crucial. The irrigation system will ultimately be

proposed with an aim of reducing aerial plant contamination. In natural

environment, it may be tricky during rainy season since the wastes may be

splashed onto the crops from soil surface. But this too has an implication since

the rainwater essentially dilutes the wastewater (which may be viewed as partial

treatment). Future experiments may need to consider these factors in order to

come up with a comprehensive and inclusive report to safeguard farmers’ use of

wastewater.

The success of wastewater unfortunately should not be viewed purely

from benefits accruing from the business. Indeed, there are other social and

cultural connotations associated with the use of this

Figure 1.1: Conceptual framework showing wastewater treatment and utilization

initiatives by different stake holders.

safeguard farmers‘ use of wastewater.

water source. The socio-cultural acceptability of produce and handling of

wastewater varies from region to region. The health effects of wastewater

production and the social and economic consequences of farmers, agricultural

laborers and their household members and consumers of wastewater-irrigated

produce have been studied in different areas (Shuval et al., 1986; van der Hoek

et al., 2002; Ennsinket al., 2003; Ensink et al., 2004). Many of the health studies,

however, lack what Mara and Cairncross called for in their well-known

“Guidelines for the Safe Use of Wastewater in” Agriculture and Aquaculture‖

(WHO, 1989), that is, ‗a thorough assessment of the local socio-cultural context.

Research conducted in urban, peri-urban and rural areas near Hyderabad city,

India, shows that such socio-economic characteristics as caste, class, ethnicity,

gender and land tenure influence the type of wastewater-dependent livelihood

activities in which each person engages (Buechler et al., 2002). The research

concluded that sale of vegetables in the wastewater-irrigated urban and peri-

urban areas was controlled by women and the venture improved their ability to

gain access to a wider variety of vegetables for themselves and for their

household members and for market. Therefore, according to Buechner et al.

(2002), recommendations based on biophysical studies that include a switch in

crops from leafy vegetables to tree crops might have ramifications for women’s

income-generating capabilities and food-security status. This is because the

introduced crops may be male dominated, hence edging out women and children

in the arena of wastewater use.

1.8 Conceptual Framework:

Figure 1.2: Conceptual framework showing wastewater treatment and utilization
initiatives by different stake holders.

Figure 1.3: Conceptual framework showing phytoremediation process and other

applications of the water hyacinth

Review of Related Literature

Wastewater Sources, Components and Collection Systems
 CHAPTER 2

REVIEW OF THE RELATED LITERATURE

2.1 Introduction

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 specific plant species like Pistia stratiotes, Water

Hyacinth, as well as Azolla pinnata to reduce toxic substances like heavy metals,

sludges, and 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

contamination of oil in Pasig River in 2017. Procedures on how to measure

bunker oil concentration is also stated.

2.2 Related Literature

2.2.1 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 affect the natural resources but also causes major strains

in the ecosystem. Interest in the use of phytoremediation as a method of solving

these 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 consists 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 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 is 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 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 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).

2.2.2 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 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).

2.2.3 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.

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, 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 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

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 bio stimulation of contaminated sites.

Azolla filiculoides removes Fe, Zn, Mn, Co, Cd, Hg, 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 many 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 petriei As
Common reed Phragmites australis Cr, Cu, Ni, Pb, S, V,
Zn, Cd
Salvinia rotundifolia Pb(II)
Salvinia natans As, Ni, Cu, Hg (II)
Salvinia minima As, Pb, Cd, Cr
Salvinia herzogii Cd, Cr
Eared watermoss Salvinia auriculata Zn, Hg, Cr
Greater duckweed, Spirodela Cu, Zn, Mn,Cr Pb
intermedia As, Hg
Spirodela polyrhiza
L.
Indian/sacred lotus Nelymbium Cr, Cu, Ba, Ti, Co, Pb
speciosum
Water spinach Ipomoea aquatica As, Cd, Pb, Hg, Cu, Zn
Eelgrass/eel weed Vallisneria spiralis L. Cu, Cd, Hg Rai

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

Miriophyllum Myriophyllum As
propinquum
Water lily Nymphaea violacea Cd, Cu, Pb, Zn
Marshwort Nymphoides Cd, Cu, Pb, Zn
germinata
Willow smartweed Persicaria Cd, Cu, Pb, Zn
lapathifolium
Fennel pondweed Potamogeton Cd, Pb, Cr, Ni, Zn, Cu
pectinatus
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
Watermilfoil, Myriophyllum Co, Cr, Cu, Pb, Zn, Ni
spicatum
Fragrant water lily Nymphaea odorata Cr
Water-milfoil Ni Myriophyllum Co, Cr, Cu, Pb, Zn,
spicatum,
Fragrant water lily Nymphaea odorata Cr
Tape grass/eel grass Vallisneria spiralis Hg
Wild celery Vallisneria Cr
americana
Shichito matgrass Cyperus Pb, Zn, Cu, Cd
malaccensis Lam.
Water pepper Polygonum As
hydropiper
Reed canary grass Phalaris Pb, Zn, Cu, Cd
arundinacea L.
Alligator weed Althernanthera As, Pb
philoxeroides
Water lettuce Pistia stratiotes As, Cr, Pb, Ag, Cd, Cu,
Hg, Ni, Zn
Floating pondweed Potamogeton Zn, Cu, Cd, Pb
natans
Willow moss Fontinalis Cu, Zn
antipyretica
Needle spikerush Eleocharis acicularis As, In, Ag, Pb, Cu, Cd,
Zn, Sb, Ni, Mg
Rigid hornwort Ceratophyllum As, Pb, Zn, Cu
demersum
Watercress Lepidium sativum L. As
Watercresses Nasturtium officinale Cu, Zn, Ni

Curly waterweed Lagarosiphon major As

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

2.2.4 Methods done in Phytoremediation

The use of plants as a phytoremediator differs on the contaminants or

pollutants to be remediated and the 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 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, 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 using 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. The increase in transpiration can

reduce contaminant migration from the site in groundwater plumes. (United

Nations Environment Programme, 2014).

2.3 Taxonomic Classification and Description of Azolla pinnata

Domain: Eukaryota Plate No. 2.1.

Kingdom: Plantae

Division: Pteridophyta

Class: Filicopsida

Order: Salviniales

Family: Salviniaceae

Genus: Azolla Verified by: John Rey C. Callado,

Museum Researcher I, National Museum
Species name: Azolla pinnata

Common name: azolla, mosquito fern, water fern

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).

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 (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, 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. 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 could 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).

2.3.1 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 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 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 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 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 and snails affect growth of Azolla by grazing on its biomass (Sadeghi,

Zarkami, Sabetraftar and Damme, 2012).

2.4 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
into a wide range of petroleum products (http//wisegeek.com/what-is-bunker-

fuel.htm, 2014).

Table 2.2 Composition of Bunker C

Ingredients CAS # Percent

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 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 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).

2.4.1 Hazardous Effects of Bunker Oil

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 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 shellfish 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).

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).

2.4.2 Bunker Oil Contamination in Pasig River

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 continuing 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 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)

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).

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

reduction is 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).

2.5 Related Studies

2.5.1 Local Studies

According to Estuaria and Viado (2013), duckweed (Lemna minor) and

water lettuce (Pistia stratiotes) can 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,

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 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 using 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 using 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 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 using 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 using 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.

2.5.2 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

freshwater ponds were removed from the ponds and introduced into each

experimental pond containing approximately 250 liters of the produced water.

Data obtained from these 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 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 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 was taken every 7 days and repeated about 4 times a

month. The results of the experiment have 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 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.

2.6 Analysis of Review of Related Literature

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, 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

freshwater fern that is considered as invasive species. It can survive and

reproduce 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.

CHAPTER 3

METHODS, MATERIALS, AND PROCEDURES

This study was conducted at the researcher’s home at Brgy. Caraycaray,

Naval, Biliran. The materials and methods used in this research are discussed in

this chapter. The researcher used the experimental method to determine whether

the Water Hyacinth can affect the sewage materials, more specifically, somewhat

clean it of certain metallic elements and other filth.

3.1 Materials Used

Water hyacinth (Eichhornia crassipes) collected from Sitio Riverside, Brgy.

Caraycaray, Naval, Biliran. The sewage (soil and water) was gotten from the

canal on the side of the Naval Highway Road near the last house from the

Riverside Residences to Naval. Other materials include cement and sand that we
had previously in stock, two plastic bags, a few small cut bamboo sticks, a short

plastic tube and two old used plastic containers (empty ice cream container) for

the sewage and plant.

3.2 Experimental Methods

In the first attempt, the sewage was fetched and batched into the plastic

container along with the Water Hyacinth, then cemented the entire plastic

container exterior except the stem and the above parts of the plant to test

whether the plant would survive if its bottom half is sealed off with only the

sewage water. The Water Hyacinth is an invasive water weed so this process will

prevent it from reproducing in the container. The samples were taken to the area

where the plant is exposed to sunlight. The whole experiment lasts for 2 weeks

and regularly checked during the course of the experiment. After 2 weeks, the

container will be opened to see what remains in its sealed portion.

The second 2-week attempt will be done in which the container is not

sealed and will be often refilled with regular water. This experiment will need

retrieve another water hyacinth and a handful of sewage soil from the same

location along with a 1 liter of sewage water. The container from the first attempt

will be used in this attempt as well. Same with the previous attempt, the plant and

container filled with sewage soil and water will be exposed to the weather

(sunlight and rain). At the last day of the experiment, the soil will be stirred to see

its murkiness if it is cleaner than when it was first brought for experimentation.
 A third attempt lasting for 11 days is planned where the bottom half of the

Water Hyacinth in the container (mainly the roots) will be sealed with a cemented

cover but this time there will be the use of plastic bags which serves as extra

sealing. In this experiment the same Water Hyacinth will be used but for the

sewage materials it will be replaced. A small tube will be placed during the

sealing process so that water can be filled in the container using that tube. Small

sticks will act as base for the cement on top of the container which will serve as

its cover. When 11 days have passed the seal will be broken off to see what has

changed with the sewage contents in the container.

3.3 Procedures

3.3.1 Attempt #1

Day 1: Obtained the plant, sewage soil where it the plant was on and sewage

water from the canal and then sealed with cement.

Day 2: Exposing the plant to sunlight starting this day and seeing if it can survive

throughout the 2 weeks of its bottom half containment.

Day 3: The plant appears to bloom its flower every early morning then closes

them at around 9 AM
Day 4: The plant appears to look rather limp on its leaves

Day 5: The stem of the plant seems to bend down as if it is weak or dying cause

not yet known

Day 9: The plant is still alive but somewhat a little bent and have not bloomed its

flowers since day 5

Day 11: The plant appears to be dying, assuming it has probably drained all of its

water
Day 12: The plant is still dying but alive and its leaves are starting to wilt. Plants

can absorb moisture with their leaves but research say that it won’t be the same

as how the roots does it.

Day 14: The last day of the experiment and time to crack open see what is left

inside. As expected, it was dying of thirst

The Water Hyacinth seems to have absorbed all the water leaving the interior dry

and it also appears to have cleaned soil during the span of 2 weeks. The plant is

still alive and wont die unless it is not given water.

The plant’s remaining soil is exposed to another small container with water and

stir it to see if the water would turn black from the soil’s content, and the result
was it just turned light brown mud colour rather than black like is Day 1. The

water content however has been absorbed by the plant leaving none to be

observed and see if it was clear or cleaned.

3.3.2 Attempt #2

Day 1: Retrieved another water hyacinth, sewage water and sewage soil from

same location and this time the container is not sealed

Day 3: Allowed the plant to be exposed to the weather in both rainy and sunny

days. The container will be allowed to get filled with rainwater.

Day 5: The experiment so far shown little signs of change but the plant mostly

absorbs water pretty fast so more water will be added when it is low

Day 6: Stirred the soil from the sewage content and it still is very murky and dark.

Changes seems to be slow, so the plant will be left alone for a couple more days.
Day 8: The plant is almost drained of water and needed to be refilled. The plant

is still healthy, and the soil seems browner but still has its grayish color. The plant

will be taken inside for the next 3 days starting the next day to see if there are

any adverse effects.

Day 13: The plant was placed outside on the morning of Day 12 and showed no

signs of wilting leaves and on Day 13 the plant nearly dries down all of its water,

so it was refilled.

These 2 other photos were taken on the same day. The water was often replaced

due to the plant absorbing so much every two days.

Day 14: On the last day of the experiment, the plant was pulled out of its

container and then the sewage soil contents were stirred, and the results still

sees the water as murky gray but slightly browner than it was on Day 1. Though I

cannot confirm it through a photo, the foul odor when I first took the sewage

materials were no longer present even as I stirred the container on this day.

The expected outcome of the experiment was incomplete due to lack of

equipment, but it was finished following the methods used in the methodology.
3.3.3 Attempt #3

Day 1: Retrieved another set of sewage material, new plastic container with no

cap/cover, the Water Hyacinth from the 2nd attempt and the sealing materials.

Placed a plastic bag on the plastic container and placed the plant which was

inside another plastic bag then sealed it with a few sticks and cement but with a

tube hole where water can be refilled into. Afterwards the cement was left to dry

off then the third experiment begins.

Day 2: The next day, the plant Day 4: This day was when the water

seems to not have received any was refilled but only by a small

damage while it was sealed from the amount (half a dipper). The plant

previous day and is doing fine isn’t showing signs of change.

 Day 5: Still no noticeable changes to the

plant on this day. The weather from the following days were mostly rainy but it

couldn’t fill as much rainwater through the tube as I thought.

Day 9: Four days of rain showers Watered down some rust filled water

have passed without being watered, from our steel faucet outside which

the plant is still alive but with one or pours slightly yellowish water for a

two wilting leaves while the other few seconds before pouring down

leaves also appears like it will follow. clear water again.

Day 10: On the next day, the plant

appears to be same as yesterday,

but it doesn’t seem to be dying any

further. As shown in the image, the

plant has these bending stems of the

leaves
Day 11: In the final day of the 3 rd experiment, it was time to open the sealed

container and witness any notable changes. The plant before opening the container

was looking healthier than on the previous day. After the cement was broken off, it

was time to unwrap the plastic bag that was holding the Water Hyacinth and sewage

materials.

The water appears to be settled down and it can be seen that the sewage soil is

noticeably brown rather than grayish in color. The soil was then stirred to see if it

was the same underneath.

The sewage content underneath also has this brown color like it is almost like regular

mud. With that, experimentation has been brought to a close for now. Future

experiments regarding whether the Water Hyacinth can really clean sewage

materials will be considered.

 CHAPTER 4
RESULTS AND DISCUSSIONS

4.1 Results

According to the results from the experiment done, the Water Hyacinth did

cleanse some of the sewage contents. The sewage water, when the debris inside

had settled down, was clear/transparent from Day 1 of each experiment attempts to

every final day of each attempt. The sewage soil turned from grayish to brownish in

color after the experiment attempts ended. The unpleasant sewage odor

disappeared after a few days. The plant survives from each experiment attempts.

Sealed containment does not affect the Water Hyacinth if given enough sustaining

water. Exposure to weather did not affect the plant and 3 three days without any

sunlight or rain did not affect it as well.

4.2 Discussions

Though the experiment was completed, it lacks data on how much of the

metallic elements and other sewage contents was removed or how much remained.
The collected sewage water only appeared murky when the sewage soil in it is

stirred or disturbed. The foul odor of the sewage did go away but it wasn’t

determined how many days it took before there was no more bad sewage odor. The

sewage soil when it was first put in the container looked so dark, murky, and full of

filth but when the experiments ended, they looked muddy brown and didn’t appear

dirty anymore. The plant does appear like it was going to wilt away on the first

attempt but that was due to lack of water when it was sealed with no orifice. The

second attempt shows that it did better due to its daily supply of fresh rusty water

which was obtained from a usually unused faucet outside the household where the

experiment was conducted. On the third attempt, the experiment needed to be

sealed off but this time with an orifice where water can be refilled inside the sealed

container which ended successfully with the sewage materials being cleaner than

when it was first brought in for this experiment and the plant still healthy which is

important so it can be used for other purposes like animal fodder/fish feed, used as

fertilizers, used to make biodegradable plastic, and a few more.

 Figure 4.1 This shows the uses of water hyacinth or how it can be utilized after water

purification

CHAPTER 5
SUMMARY, CONCLUSION, AND RECOMMENDATIONS

This chapter summarizes the results of this study and the conclusions drawn

from the experiment as well as the researcher’s recommendations for future studies.

5.1 Summary of Findings

 Water hyacinth can be used to treat sewage with effluent standards. It is

highly suitable for tropical wet and dry climate. Also, requires minimal space for the

set-up and if the wastewater flow is high, then a series or parallel set-up of water

hyacinth can be done, thus using land and space to the maximum extent while also

sealing off the top of the containment. The plant doesn’t require any

energy/electricity for its function and hence it is suitable where there isn’t proper

supply of power or where the cost of energy is too high. The plant is susceptible to

dying very fast usually in 1 day when there is no water during its containment but can

very well be left alone to do its role if sealed containment has flowing supply of

water.

5.2 Conclusions

Although water hyacinth generally occurs in places with little to no flow such

as lakes and canals, it also thrives in the tidally influenced ecosystems in shallow

water habitats and other low-velocity areas. These hydrologic conditions create a set

of habitat conditions that is different than other systems where water hyacinth effects

on water quality have been extensively studied. The tidal hydrology moves water in

and out of water hyacinth patches, such that effects of water hyacinth on water

quality can readily be exported to surrounding areas. This process can be done as

constructed underground water systems in which movement of the plant or just the

purified water to these surrounding areas is possible. In some study, dissolved

oxygen and turbidity became more similar to regional averages after treatment,

which suggests removing water hyacinth may return habitat and water quality values

to their pre-infestation state. During the three times of experimentation on the water
hyacinth, the plant did indeed survive sealed containment which could lead to other

procedures to involve sealing half of the plants’ body to prevent unwanted

proliferation. A comprehensive water quality data collection, additional information on

the success of herbicide treatment, and studies that focus on changes to fish

assemblages are crucial to informing management about the effect of water hyacinth

and its control efforts on the ecosystem, including species of concern.

5.3 Recommendations

Based on these findings, the study discovered some of the recommendations

that can be of use both to the future studies and to the community of the area of

study.

To begin with the government and NGOs should come in and encourage the

residents not only to rely on fishing activities but also embrace the plant as of

economic importance. This can be achieved if the government can establish cottage

industries that can harvest the plant and use its use in processing of board are

sufficiently good for use on indoor partition walls and ceilings. The serious damage

of the ecological system of the area around the lake caused by water hyacinth can

be controlled by using the plant in large-scale production of charcoal briquettes from

water hyacinth as discovered by Eden (1994). The possibility of converting water

hyacinth to biogas should also be embraced. Conversion of other organic matter,

usually animal or human waste, is a well-established small and medium scale

technology in a number of developing countries, notably in China and India. The

government should encourage the consumption of the perceived less economically

important fish species and encourage value addition technics to increase their

economic viability

The water purification of water hyacinth still needs to be studied whether it

could effectively eliminate many of the toxic contents of the sewage materials from

different canals using proper testing equipment done in a laboratory. Containment

procedures used for preventing or even stopping the plant’s fast proliferation should

also be tested in a large body of water to also solve the clogging problems caused

by the plant’s roots and overgrowth. Utilizing the plant while testing them would

hasten the process if the government and NGOs applies this to the community of

people living near lakes, rivers, and/or farmlands to realize the constructed wetlands.

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